Search

The aim of this study is to present development of tissue engineering and clinical application of cultured epidermal autografts (CEA). Advanced therapy medicinal products (ATMPs) are medicines for humans that include gene, somatic cell and tissue-engineered therapeutic products. Cultured keratinocytes regenerate epithelium and belong to the category of ATMP as tissue-engineered products. The development of ATMPs in Croatia began during 2002 in collaboration with the Ruđer Bošković Institute, the Clinic of Traumatology and the Children's Hospital Zagreb on the project Production of skin grafts in vitro. Engineering Laboratory was built in May 2005 in accordance with Good Manufacturing Practice and clean room technology. Tissue and Cell Bank (TCB) was established during 2007. The procedure includes isolation of keratinocytes from the skin biopsy (about 4-6 cm2/0.3 mm thick) after which they are seeded onto a feeder layer of 3T3 cells and incubated at 37 °C, 5 % CO2. Preparation of the optimal number of grafts is accomplished within 3- 4 weeks depending on the area of the injury. Immunocytochemistry (ICC) combines histological, immunological and biochemical techniques in the identification of specific tissue components. Using flow cytometry enabled high-throughput analysis of keratinocytes and residual feeder cells. Detection of bacterial endotoxins was determined using a Lysate of Amebocyte Limulus test. According to the EMA guidance the recommended endotoxin limit for release testing is ≤ 0.5 EU/mL (endotoxin units). Cell growth was monitored on a daily basis on microscope and analyzed on 14th day for evaluation of colony- forming efficiency (CFE). CFE was calculated as the number of colonies divided by the total number of seeded cells (1000 or 1500) and multiplied by 100 to express the result as a percentage. Quality control involves potency (yield, viability, CFE), purity (ΔNp63, CK AE1/AE3, CK5/6, CK14, CK19, vimentin), impurity (remaining 3T3 cells) and safety (sterility, mycoplasma, bacterial endotoxins).The first successful production of epidermal grafts began in the Clinic of Traumatology in September 2002 with 10 epidermal grafts of 700 cm2. This retrospective analysis spans a period from February 2002 to October 2003. The project included donors (n=15), from 2 to 66 years old with total 92 CEA. Microbiological control has proven the sterility of all keratinocytes, cell media as well as epidermal transplants. From July 2007 to March 2022 in TCB, donors (n=62) were from 1 to 74 years old with total 2235 CEA from which 89.2 % were transplanted and 10.8 % discarded. The most common reasons for discardment were patient’s death, initial microbiological contamination (Acinetobacter baumannii, Cutibacterium acnes, Micrococcus spp., Pseudomonas aeruginosa, Staphylococcus capitis, Staphylococus epidermidis and other coagulase negative staphylococcus, etc.) and technical reasons (problem with feeder layer and decontamination of skin biopsy). Growth media from CEA was monitored for bacterial endotoxins prior to clinical application. Our results were < 0.125 EU/mL. CFE for 1000 cells was ≈ 9, 3 % and for 1500 cells was 8.3-15.1 %. ICC analysis showed 100 % epithelial cell marker CKAE1/AE3 positive cells (A), 100 % CK14 expressed in mitotically active basal layer cells (B), without CK19 positive differentiated keratinocytes (C), with 22.5 % proliferation marker Ki-67 positive cells (D) and 32 % vimentin positive cells represent pool of good quality of keratinocytes. Flow cytometry analysis shows high percentage of ΔNp63 (99.6 %) positive cells and low percentage of anti-feeder cells (1.66 %) positive cells ensure a good quality of CEA for the transplantation. Keratinocytes prepared as epidermal grafts or suspension with fibrin glue contributed to the survival of severely burned patients.

Spongy bone is used in reconstructive procedures as a valuable material for bone regeneration and replacement. The aim was to examine the aseptic procurement and cutting of femoral head fragments in the operating room, the sterility of spongy bone obtained in the microbiological safety cabinet (MSC) and to show histological structure and vitality of tissue. During the implantation of a total hip endoprosthesis in living donors, the femoral heads were removed and cut into fragments. A swab for microbiological and a biopsy for pathohistological analysis were sampled from each head. Fragments were processed in MSC and the sterility of spongiosis was controlled by analysing duplicate swabs and biopsies by direct inoculation in a liquid medium for anaerobic and aerobic bacteria and fungi. Sterility of environmental conditions (surface, air and operator’s fingers) was controlled by contact and sediment plates. 27 samples were microbiologically analysed: 16 from the operating room (biological n=10, environmental n=6) and 11 from MSC (biological n=7, environmental n=4). A 100 % sterile result of bone tissue and operator's fingers was obtained in the operating room and the results of air control were within acceptable limits. All results sampled in the MSC were 100 % sterile. Pathohistological analysis confirmed vital osteocytes in the lacunae of the circumferential lamellae, individual osteoclasts and numerous osteoblasts in the surface of the bone without any pathological changes.

UVOD: Unazad 15 godina jedna od osnovnih djelatnosti Banke tkiva i stanica je prikupljanje koštanih presadaka alogenih darivatelja (glave bedrene kosti). Koštani presaci se uzimaju tijekom operativnog zahvata ugradnje TEP-a kuka. Tijekom zahvata operater procjenjuje kvalitetu koštanog presatka i donosi konačnu odluku o pohrani tkiva. Prilikom uzimanja, u operacijskoj sali, svim koštanim presacima uzimaju se uzorci za mikrobiološku kontrolu. Presaci se pohranjuju i čuvaju u zamrzivačima banke na temperaturi -80 °C s rokom trajanja 5 godina. Od 2012. godine u suradnji s Klinikom za ženske bolesti i porodništvo KBC Sestre milosrdnice, razvijena je i djelatnost prikupljanja posteljica i pripreme amnijskih presadaka. Posteljice se uzimaju isključivo tijekom poroda carskim rezom od alogenih darivateljica. Nakon uzimanja, daljnja obrada posteljice provodi se u čistim prostorima (prostori s najvišim stupnjem mikrobiološke čistoće) koji se nalaze u Klinici za traumatologjiu. Od plodnih ovoja se odvaja amnijska membrana koja prolazi proces dekontaminacije te se reže na presatke različitih dimenzija. Presaci amnijske membrane čuvaju se u krioprezervansu u zamrzivačima na temperaturi -80 °C s rokom trajanja 2 godine. Svi darivatelji prije darivanja obavezno prolaze kroz strogi proces odabira i eliminacije te se nakon darivanja testiraju na krvlju prenosive bolesti kako je propisano nacionalnim i europskim zakonskim propisima. REZULTATI: Koštani presaci se koriste u rekonstruktivnim zahvatima ortopedske kirurgije u slučajevima kada se autotransplantatom ili umjetnim materijalom ne mogu postići željeni rezultati. Amnijski presaci se najčešće koriste u oftalmologiji za liječenje ozljeda rožnice. Unazad nekoliko godina počeli su se koristiti i za liječenje ozljeda kože (npr. opekline, avulzije kože i rane koje teško cijele). Svi primatelji presadaka se prate nakon primjene radi uvida u ishod liječenja i prihvaćanja transplantata. ZAKLJUČAK: Tijekom dugogodišnjeg praćenja rezultata liječenja navedenim presacima nije zabilježena niti jedna neželjena reakcija kod primatelja presadaka. Zbog navedenog zaključujemo da su naši presaci kvalitetni i sigurni za kliničku primjenu.

Transplantacija osteohondralnog alografta tehnika je kojom se može tretirati širok spektar patologije koja uključuje hrskavicu i podležeću kost. Tehnika je posebno pogodna za mlađu populaciju pacijenata sa značajnim (> 2.5 cm2) osteohondralnim defektom, kao primarni ili kao revizijski zahvat. Predstavljamo slučaj 28- godišnjeg pacijenta koji se prezentirao dugogodišnjom boli u području lateralnog kompartmenta desnog koljena, najizraženijom prilikom fleksije. Učinjenom radiološkom obradom nađena je osteohondralna lezija dimenzija 3x2 cm u području lateralnog kondila femura koja je shvaćena kao nestabilni OCD. Učinjen je pokušaj fiksacije s dva chondral darta. Nakon inicijalnog poboljšanja dolazi do pogoršanja stanja te je pacijent 8 mjeseci kasnije podvrgnut operativnom zahvatu u vidu artroskopske toalete te mikrofraktura. Nakon drugog zahvata izostaje adekvatan klinički odgovor. Naposljetku se odlučujemo za kirurški zahvat transplantacije osteohondralnog alografta. U suradnji s Bankom tkiva Klinike za traumatologiju te Bankom tkiva i kosti Barcelona Španjolska pribavlja se smrznuti alograft lateralnog kondila femura. Korišten je Arthrex BioUni® OATS® instrumentarij. Zbog tehničkih uvjeta nije bilo moguće osigurati originalnu Arthrex radnu stanicu te dizajniramo vlastitu radnu stanicu za fiksaciju alografta tokom obrade koja se temelji na modifikaciji standardnog Ilizarov okvira. Operativni zahvat je učinjen u spinalnoj anesteziji. Izmjerena je veličina defekta te je prema mjeri obrađen alograft fiksiran na radnoj stanici. Zatim je učinjena transplantacija grafta. Operativni zahvat i postoperativni tijek protekao je uredno. Šest tjedana nakon zahvata radiološki su vidljivi znakovi koštanog preraštavanja, bez znakova odbacivanja implantata. Pacijent je subjektivno bez tegoba, urednog opsega pokreta. Zaključak prikaza slučaja je da je transplantacija osteohondralnog alografta operacija s izvrsnim ranim rezultatima za velike osteohondralne defekte u mlađoj populaciji. Također, naša radna stanica može biti izrađena uz pomoć materijala koji se nalaze u inventaru svakog traumatološkog odjela bolnice te je prikladna alternativa komercijalno dostupnom setu.

UVOD: Banka tkiva i stanica djeluje u Klinici za traumatologiju od 2005., a od rujna 2019. godine je dio Zavoda za transfuzijsku i regenerativnu medicinu KBC Sestre milosrdnice. Osnovne djelatnosti banke su tkivno bankarstvo (prikupljanje, uzimanje, očuvanje, pohrana i raspodjela glava bedrene kosti uzetih od živih alogenih darivatelja i amnijskih presadaka pripremljenih od posteljica živih alogenih darivateljica) i tkivno inženjerstvo (proizvodnja lijekova za naprednu terapiju, engl. Advanced Therapy Medicinal Products – ATMP), tj. uzgoj keratinocita i limbalnih stanica in vitro za autolognu primjenu. REZULTATI: Glava bedrene kosti uzima se od živih darivatelja tijekom operacije ugradnje TEP-a kuka.. Provodi se detaljan pregled darivatelja, mikrobiološka analiza brisa glave koji se uzima tijekom operacijskog zahvata prije pohrane u sterilnu posudu te analiza krvi darivatelja na krvlju prenosive bolesti. U tablici 1. je plavom bojom prikazan broj prikupljenih presadaka, crvenom broj presadaka za kliničku primjenu, a zelenom broj primijenjenih presadaka godišnje od 2007. do 2021. Do 2020. najčešći razlog uništenja presadaka je bio pozitivan mikrobiološki nalaz brisa, a unazad dvije godine i neprikladnost darivatelja uočena nakon darivanja. Amnijski presaci se proizvode od posteljica živih darivateljica uzetih tijekom carskog reza.. Također se provodi detaljan pregled darivateljica, mikrobiološke i histološke analize tkiva te analiza krvi. U tablici 2. je plavom bojom prikazan broj proizvedenih presadaka, crvenom broj presadak za kliničku primjenu, a zelenom broj primijenjenih presadaka godišnje od 2012. do 2021. Spongioza glave bedrene kosti se koristi kod koštanih defekata dok se amnijski presaci koriste kod teških ozljeda rožnice i kože. Uzgoj autolognih keratinocita i limbalnih stanica in vitro provodi se u čistim sobama. Keratinociti se primijenjuju kodopeklina drugog i trećeg stupnja velike tjelesne površine, a limbalni presaci kod potpunog deficita limbalnih stanica. ZAKLJUČAK: Regenerativna medicina predstavlja budućnost medicine u obnovi strukture i funkcije teško oštećenih tkiva i organa.

Na kongresu su prikazani naši rezultati kod transplantacije uzgojenih autolognih limbalnih stanica u liječenju kombustije oka

Cultivation of limbal stem cells (LSC) is a well-established method of cellular therapy for patients with limbal epithelial stem cell deficiency caused by chemical burns, thermal injuries or chronic immune inflammatory diseases. Limbal cells were obtained from ocular biopsies cultivated on a feeder-layer made from 3T3 cells. This cell therapy was classified as advanced therapy medicinal products (ATMPs).In the Laboratory of Tissue Engineering in microbiological cabinet (which is clean room class A) were we prepared limbal stem cell grafts. With methods of flow cytometry it is possible to detect anti-feeder layer positive cells, stem cells and differentiation cells.Haematoxylin and eosin stained histological images showing firstly cell morphology and secondly stratification of limbal grafts. Immunohistochemistry analysis detected positive staining of stem cells (p63α), Ki67 and CK3. Live-cell imaging of human LSC were obtained in collaboration with Ruđer Bošković Institute, Zagreb. The cell forms a spindle, a molecular machine responsible for equal distribution of chromosomes between the daughter cells. It is recommended to use reagents, growth medium and water certified to contain endotoxin in minimal concentration. Cultivated limbal epithelial transplantation (CLET) has shown to be an effective therapy for LSCD, with clinical trials reporting an average success rate of 70 to 80%

Tyrosinemia type 1 is an autosomal recessive aminoacidopathy caused by fumarylacetoacetate hydrolase (FAH) deficiency. Consequently, tyrosine and its metabolites accumulate, resulting in liver and kidney toxicity. Symptoms of the disease usually manifest after three weeks of life and include vomiting, failure to thrive, hepatomegaly, jaundice, bleeding diathesis, rickets and renal tubular dysfunction. Untreated, the disease eventually progresses to liver or kidney failure and generally results in a fatal outcome. Expedient diagnosis is critical because an early start of treatment can increase the likelihood of a positive outcome. Here, we report on a male newborn with a family history positive for tyrosinemia type 1 who was subjected to a metabolic work-up immediately after birth. Amino acids were quantified by tandem mass spectrometry coupled with ultra performance liquid chromatography. Urinary organic acids were analyzed on capillary gas chromatography coupled with mass spectrometry. DNA analysis of the FAH gene was performed by Sanger sequencing. On the first day of life, the patient's plasma amino acids showed an increased tyrosine concentration, while urine organic acids detected succinylacetone, a tyrosine metabolite specific for tyrosinemia type 1. The patient's DNA analysis revealed homozygosity of the c.554-1G > T mutation in the FAH gene, which was consistent with the diagnosis. Nitisinone treatment, combined with a dietary restriction of tyrosine and phenylalanine, was introduced immediately. Regular visits and measurement of amino acid concentrations, which enables therapy adjustment and treatment efficiency monitoring in patients with tyrosinemia type 1, has continued over the past 4+ years, and is expected to continue.

Novorođenački probir je u Hrvatskoj od 1986. godine obavezna mjera zdravstvene zaštite novorođenčeta. Krajem 2017. godine uvođenjem tehnologije tandemske spektrometrije masa program novorođenačkog probira je proširen, uz probir na fenilketonuriju i prirođenu hipotireozu na još šest rijetkih nasljednih metaboličkih poremećaja. Cilj je ovog članka ukazati na kompleksnost programa kao i na značajnu ulogu medicinskih sestara u njegovom provođenju. Opširnije su opisani mogući prijeanalitički čimbenici koji utječu na rezultate mjerenja kao i na kvalitetu novorođenačkog probira.

Kontinuirano uvođenje novih dijagnostičkih metoda i novih biomarkera uglavnom podrazumijevaju nove i složene laboratorijske procese. Otvoreni analitički sustavi poput elektroforetskih i kromatografskih sustava omogućavaju razvoj novih postupaka te uvođenje novih, klinički relevantnih biomarkera u različita klinička područja. Poznavanje takvih tehnika i fizikalno-kemijskih načela na kojima se te tehnike temelje osigurava svrhovitost, tj. stvarnu potrtrebu za odgovarajućim stručnjacima.

Rijetke bolesti etiološki su heterogena skupina najčešće nasljednih, kroničnih i degenerativnih bolesti s pojavnošću manjom od 5 na 10 000 osoba. Poznato je više od 7000 rijetkih bolesti od kojih više od 600 ima osobitosti nasljednih metaboličkih poremećaja. Zbog velike kliničke heterogenosti i zahtjevnih dijagnostičkih testova put do konačne dijagnoze je često dug te uključuje i obradu u inozemnim laboratorijima. Unatoč tome, dio bolesnika ostaje bez konačne dijagnoze. U situacijama kada je bolesnik vitalno ugrožen uz mogućnost letalnog ishoda i pri sumnji na nasljednu metaboličku bolest, biopsija kože je dio standardnog postupka. Posljednjih godina sve je više dostupnih mogućnosti liječenja za širok spektar nasljednih metaboličkih bolesti. Osnovni preduvjet za njihovu djelotvornost je pravovremena dijagnoza. Kultivirani kožni fibroblasti pohranjeni u biobanke, vrijedan su biološki materijal. Osim za postavljanje dijagnoze, mogu se koristiti i za provjeru djelotvornosti novih pristupa liječenju kao što su male molekule pratitelji. Sve navedeno upućuje na značaj organiziranja biobanke fibroblasta prema međunarodno prihvaćenim standardima.

Introduction Tyrosinemia type 1 is an autosomal recessive aminoacidopathy caused by deficiency of fumarylacetoacetate hydrolase (FAH), an enzyme responsible for the final step of tyrosine breakdown. Consequently, tyrosine and its metabolites accumulate, resulting in toxicity to liver and renal cells. Symptoms of the disease begin usually after three weeks of life and include vomiting, failure to thrive, hepatomegaly, jaundice, tendency to bleed and bruise easily, rickets and renal tubular dysfunction. The untreated disease eventually progresses to liver or kidney failure and usually ends fatally in early childhood. In patients who survive, there is an increased risk of hepatocellular carcinoma. Prompt diagnosis is extremely important because early start of treatment may prevent fatal outcome. Subjects and methods A male newborn with positive family history of tyrosinemia type 1 was subjected to a metabolic work-up immediately after birth. Amino acids were quantified by tandem mass spectrometry coupled with high performance liquid chromatography, LC-MS/MS (API 3200, Sciex ; UPLC Nexera, Shimadzu). The results were obtained using aTRAQ™ reagent by Sciex with internal standards of known concentration for each amino acid. DNA analysis of the FAH gene was performed in Sahlgrenska University Hospital in Gothenburg, Sweden. Results At first day of life, patient's plasma amino acid analysis showed increased concentration of tyrosine (169 µmol/L ; normal: 42-135 µmol/L) while urine organic acid analysis detected succinylacetone, a tyrosine metabolite specific for tyrosinemia type 1. The diagnosis was confirmed by patient's DNA sequencing, which revealed a homozygosity for the c.554-1G>T mutation in the FAH gene. As soon as the diagnosis was established, nitisinone treatment, combined with a dietary restriction of tyrosine and phenylalanine, was introduced. Routine measurement of amino acid concentrations continued while a patient has been regularly monitored. Conclusion Regular measurement of the concentration of plasma amino acids using LC-MS/MS enables therapy adjustment and treatment efficiency monitoring in patients with tyrosinemia type 1.

Introduction Gyrate atrophy of the choroid and retina (OMIM: 258870) is a rare autosomal recessive disease that occurs due to the lack of the mitochondrial enzyme ornithine aminotransferase (OAT ; EC 2.6.1.13). Hyperornithinemia causes degeneration of the retina with the symptoms of disease like myopia, reduction in night vision and progressive resolution of vision loss. Our patient is an 8- year-old girl with impaired vision and strabismus. First symptoms appeared at the age of four years. Further ophthalmic examination revealed retinal dystrophy and macular edema in both eyes, with numerous circular sharply limited zones in the retina. The characteristic ophthalmologic finding was suspected on deficiency of an enzyme OAT involved in the metabolism of the amino acid ornithine. Materials and methods Amino acid analysis was performed using high-performance liquid chromatography coupled with electrospray tandem mass spectrometry LC-MS/MS (API 3200, Sciex ; UPLC Nexera, Shimadzu). Using aTRAQ™ kit for physiological amino acid analysis allows quantitation of free amino acids in plasma. Analyst and Cliqid software applications (Sciex) were used to obtain data for relative and absolute quantitation of up to 45 amino acids. Results As part of the metabolic check-up, analysis of amino acids in plasma revealed significantly increased concentration of ornithine (1039 µmol/L ; normal: 20-155 µmol/L). Molecular genetic analysis detected the homozygous mutation in exon 7 of the OAT gene that has not been described as yet (c.868_870delCTT p.Leu290del ; Zurich Children's Hospital). The success of low-protein diet and response to pyridoxine (initial dose of 500 mg with poor response) was monitored by measuring ornithine concentration for more than one year. Plasma ornithine levels decreased by 30-40% (fell to 608 µmol/L). Our expectation from including a recent therapy with L-lysine is to reduce reabsorption of arginine and ornithine in the kidney tubules. Conclusion Hyperornithinemia is the main pathogenic factor in gyrate atrophy and can be influenced by diet. LC-MS/MS is one of the best methods for diagnosing, treatment and follow- up of hyperornithinemia.

Background: Glutaric aciduria type 3 (GA3) is an autosomal recessive disorder caused by mitochondrial succinate-hydroxymethylglutarate CoA-transferase deficiency. It is characterised by increased persistent, isolated excretion of glutaric acid. There is no distinctive phenotype associated with this disorder. The diagnosis is established by excluding other causes of glutaric aciduria and by gene analysis. Case report: Patient 1 presented at the age of 15 months with recurrent vomiting, hypotonia, ataxia, left abducens paresis and symmetrical patchy white matter changes on brain MRI mimicking postvaccinal encephalopathy. She recovered completely. Patient 2 experienced episodes of agitation and unresponsiveness lasting 2 hours during 3 consecutive nights at the age of 12 months. At the age of 3 years he had signs of attention deficit hyperactivity disorder. In both families parents were unrelated. Results: Organic acid analysis showed elevated urinary excretion of glutaric acid in both patients ranging from 39-98 mmol/mol of creatinine in patient 1 and from 90-133 mmol/mol of creatinine in patient 2 (NT (p.Arg336Trp) mutation in a homozygous state in both patients. Discussion: Our cases suggest that under certain circumstances GA3 may not be a benign condition, as indicated by encephalopathic crises experienced by the first reported patient more than 20 years ago after the first description. The c.1006C>T mutation is known to be causing GA3. Its appearance on four unrelated alleles in our patients points to its high frequency in different populations and possible hot spot of the SUGCT gene. More information and additional haplotype analysis are needed for a more accurate conclusion.

Određivanjem koncentracije slobodnog karnitina i profila acil karnitina tehnologijom tandemske spektrometrije masa moguće je detektirati veliki broj nasljednih metaboličkih bolesti (poremećaji beta oksidacije masnih kiselina i organske acidemije). Istaknute su prednosti i nedostaci različitih vrsta uzoraka (suhe kapi krvi i plazme). U dugoročnom planu je uvođenje analize profila acil karnitina iz plazme što bi u konačnici doprinijelo povećanju specifičnosti u dijagnostici nasljednih metaboličkih bolesti.

Citrulinemija tip 1 rijedak je autosomno recesivni poremećaj uzrokovan nedostatkom enzima argininosukcinat sintetaze. Nedostatak ovog enzima uzrokuje nakupljanje amonijaka u krvi i drugim tjelesnim tekućinama. Nakupljanje amonijaka dovodi do razvoja simptoma kao što su povraćanje, smanjeni apetit, letargija, napadaji, hipotonija, respiratorni distres, cerebralni edem i abnormalno povećanje jetre, a može uzrokovati i komu. U slučaju iznimno visokih vrijednosti amonijaka nužno je započeti s odgovarajućim medicinskim liječenjem kako bi se spriječila koma i njome izazvana popratna neurološka oštećenja. Sastavni dio liječenja je odgovarajuća dijetoterapija koja za cilj ima smanjeni unos proteina (niskoproteinska dijeta) kako bi se spriječilo nakupljanje amonijaka. Simptomi bolesti mogu se javiti u neonatalno doba (klasična citrulinemija) ili u kasnijim fazama života (blaži oblik), npr. za vrijeme ili nakon trudnoće. Neke žene ostaju nedijagnosticirane do akutnog pogoršanja simptoma tijekom trudnoće ili u razdoblju nakon porođaja. Ne postoje čvrste preporuke za liječenje kod žena kojima je bolest dijagnosticirana tijekom trudnoće. Cilj ovoga rada je prikazati utjecaj dijetoterapije na ishod trudnoće kod novodijagnosticirane citrulinemije tipa 1. Prikazan je slučaj žene u dobi od 20 godina, koja je hospitalizirana u 27. tjednu trudnoće zbog zatajenja jetre i encefalopatije. Provedena je metabolička obrada i obzirom na visoke vrijednosti amonijaka (220 μmol/L), citrulina (921 μmol/L ; N 10-55 μmol/L) i glutamina (1141 μmol/L ; N 340-740 μmol/L) u plazmi te nemjerljivo niske razine arginina i argininosukcinata postavljena je sumnja na citrulinemiju tipa 1, koja je kasnije genetskom analizom i potvrđena. Uvedena je odgovarajuća dijetoterapija sa smanjenim unosom proteina i odgovarajućim kalorijskim unosom uz nadoknadu arginina i natrijevog benzoata što je rezultiralo potpunim oporavkom. Unos proteina se povećavao kako je trudnoća odmicala, uz stalno praćenje liječnika i dijetetičara. Bolesnica je pred porod podnosila unos proteina od 1, 1 g/kg TM. Neposredno pred porod ponovno je uvedena niskoproteinska dijeta. U 39. tjednu rodila je zdravo dijete. Unos proteina postepeno se povećavao do 1, 1 g/kg TM, uz održavanje odgovarajućih vrijednosti amonijaka i aminokiselina u plazmi, te se pretpostavlja da bi bolesnica podnosila i viši unos. Možemo zaključiti da je odgovarajuća dijetoterapija esencijalan dio liječenja trudnica s citrulinemijom tipa 1 i da doprinosi pozitivnom ishodu i za majku i za dijete

Uvod: Sindrom MELAS (mitochondrial encephalomyopathy, lactacidosis and stroke-like episodes) je jedna od najčešćih maternalno nasljednih mitohondriopatija. Najčešće je uzrokovana mutacijom m.3243A>G gena MT-TL1 koja dovodi do poremećenog stvaranja mitohondrijskih proteina i narušenog energetskog metabolizma. Klinička slika obuhvaća širok spektar simptoma i vrlo varijabilan tijek (zbog heteroplazmije i „efekta praga“). Najčešće se simptomi javljaju u drugom desetljeću života kao migrenozne glavobolje, epileptički napadi, epizode slične moždanom udaru, povraćanje, mišićna slabost, slabljenje vida i sluha, nizak rast, kardiomiopatija, i dr. Biokemijsko obilježje i putokaz k dijagnozi je povišen laktat u krvi i likvoru, no nije uvijek prisutan. Prikaz bolesnika: Desetogodišnji dječak je tijekom virusne bolesti razvio jaku glavobolju, povraćanje, poremećaj svijesti i žarišne, unilateralne napade. Anamnestički se doznalo da majka boluje od sindroma MELAS. Učinjeni su CT mozga koji je pokazao edem desno okcipitalno i lumbalna punkcija kojom je nađen povišen laktat (6, 30 mmol/L, N G. Dječak je otpušten s peroralnim L-argininom u terapiji. Drugu epizodu je imao nakon par mjeseci, praćenu glavoboljom, povraćanjem, poremećajem svijesti i laktacidozom. MR mozga je pokazao potpunu regresiju prijašnje i novu leziju sa suprotne strane. Na primjenu intravenskog arginina, odgovarajući parenteralni kalorijski unos i potporne mjere dolazi do potpunog oporavka. Rasprava i zaključak: Epizode slične moždanom udaru su odraz regionalnog poremećaja perfuzije mozga uslijed mikroangiopatije i manjka dušičnog oksida. Nužno ih je pravovremeno prepoznati i liječiti analgeticima, antipireticima, održavanjem adekvatne hidracije i kalorijskog unosa infuzijom glukoze, po potrebi lipida, te intravenskom infuzijom arginina koji ima vazodilatatacijski učinak. Doza koja se najčešće preporučuje je 500 mg/kg. Potrebno je korigirati hipoglikemiju, elektrolitski disbalans i hipotenziju, te liječiti epileptičke napade. Može se dati tiamin. Uz navedene mjere moguće je zaustaviti daljnje širenje lezije i spriječiti/umanjiti trajne posljedice. Trajna terapija uključuje peroralni L-arginin u dozi od 200 mg/kg na dan, za koji se dokazalo da smanjuje broj i težinu novih epizoda sličnih moždanom udaru, ili odnedavno citrulin. Nužno je izbjegavati lijekove i navike koji djeluju toksično na mitohondrij, stres i iscrpljivanje.

Infantilni oblik PB je autosomno recesivno nasljedni manjak kisele alfa-glukozidaze (GAA) s ostatnom enzimskom aktivnošću 3 sekunde, hipotenzivno 60/40 mmHg, s hepatomegalijom, oligurično, teško generalizirano hipotonično. Miješana venska saturacija 20-30%, laktati 3-4 mmol/L, NT-proBNP >35000 pg/mL, povišene aktivnosti AST 162-300 U/L, ALT 102-195 U/L, LD 1106-1996 U/L, CK 731-1204 U/L. Na RTG-u bila je izrazita kardiomegalija ; EKG-u ekstremna hipertrofija lijeve klijetke ; UZV-u ekstremno zadebljan miokard (septum 18, stražnja stijenka 20 mm) oslabljene kontraktilnosti (EF 15-20%) i rastezljivosti. Aktivnost GAA u limfocitima bila je nemjerljivo niska, a CRIM status negativan u skladu s homozigotnom mutacijom gena GAA (c.2269C>T, p.Q757X). Dijete je zbrinuto intubacijom, mehaničkom ventilacijom, dobutaminom, milrinonom, dopaminom, ultrafiltracijom te ostalim lijekovima i mjerama intenzivnog liječenja. U jednom navratu je oživljavano uz masažu srca i defibrilaciju kroz 4 minute. Prema protokolu za CRIM-negativne bolesnike započeta je imunomodulacija (rituksimab, metotreksat, IVIG) kako bi se postigla imunotolerancija i poboljšao inače loš ishod CRIM-negativnih bolesnika. Započeto je specifično liječenje nadomještanjem enzima rGAA. Nakon 3 mjeseca liječenja dijete je svijesno, bistro, traheotomirano, diše uz tlačnu potporu, cirkulacijski stabilno, hipotonično, motorički značajno zaostalo. NT-proBNP je značajno niži (7642 pg/mL), aktivnosti mišićnih enzima su u padu, značajno je manja kardiomegalija, stražnja stijenka miokarda tanja (11-12 mm), EF dvostruko viša (35-40%). Tijek bolesti u naše bolesnice pokazuje važnost rane dijagnoze za ishod liječenja, koji je u nje vrlo upitan, bez obzira na poboljšanje stanja srca.

UVOD: U lizosomske bolesti nakupljanja ubraja se više od 50 rijetkih, multisistemskih, nasljednih, metaboličkih bolesti uzrokovanih manjkom lizosomskih enzima koji sudjeluju u razgradnji brojnih organskih makromolekula. Neke od ovih bolesti se mogu uspješno liječiti ukoliko su pravovremeno prepoznate i započeta terapija prije nastanka ireverzibilnih promjena. Uzorak suhe kapi krvi na filtar papiru (engl. Dried Blood Spot, DBS), osim za novorođenački probir, ima sve veću primjenu u selektivnom probiru lizosomskih bolesti nakupljanja. Glavne prednosti DBS: mali volumen uzorka ; jednostavna priprema, pohrana i dostava uzoraka (stabilnost analita kroz duži vremenski period) ; primjenjivost za brojne laboratorijske tehnike. Predanalitičke pogreške koje nastaju izvan laboratorija utječu na točnost nalaza jer smanjuju osjetljivost i specifičnost laboratorijskog postupka . Predanalitički čimbenici za uzorak DBS jesu homogenost filtar papira, rukovanje i pohrana filtar papira (vlažnost), volumen krvi nanesen na filtar papir, vrijeme apsorpcije krvi, hematokrit. MATERIJALI I METODE: U periodu 2013.-2016. primljeno je iz suradnih ustanova 3605 uzoraka DBS (kapilarna krv ; venska krv ; filtar papir Whatman 903®) ; uz to u metaboličkom laboratoriju pripremljeno je 1340 uzoraka DBS (EDTA-venska krv, standardizirani volumen krvi, filtar papir Whatman 903®) bolesnika s kliničkom sumnjom na lizosomsku bolest nakupljanja (Fabrijeva bolest, Pompeova bolest, Gaucherova bolest, Mukopolisaharidoza tipa I). Aktivnosti lizosomskih enzima mjerene su “in house” fluorimetrijskim metodama uz sintetske supstrate obilježene 4-metilumbeliferonom (spektrofluorimetar Varian Cary Eclipse). Kvaliteta uzorka DBS ispitana je mjerenjem aktivnosti kontrolnog enzima. REZULTATI: Predanalitičke pogreške radi kojih je zatraženo ponavljanje uzorkovanja krvi , nađene su kod uzoraka poslanih iz suradnih ustanova (nepravilno nanašanje krvi na filtar papir, nedostatno sušenje kapi krvi, nepropisna pohrana i prijenos uzoraka. β-glukozidaza (n=2327 ; 12%), α-glukozidaza (n=2799 ; 22%), α-galaktozidaza (n=1998 ; 17%), α-L-iduronidaza (n=1135 ; 18%. Kod uzoraka DBS priređenih u metaboličkom laboratoriju nisu nađene predanalitičke pogreške.

Purpose: To present the clinical course, diagnostic and treatments of six-year-old girl with gyrate atrophy and bilateral cystoid macular edema. Methods: Case report. Four-year-old girl was found with low vision and convergent strabismus in a systematic medical examination. After 2 years of amblyopia treatment bilateral macular edema was detected and the child was referred to The Department of Ophthalmology. Results: A six-year-old girl, status at presentation - both eyes: BCVA 0.15, cystoid macular edema, sharply demarcated oval zones of peripheral choroidal atrophy. Antiedematous therapy with systemic corticosteroids and topical carbonic anhydrase inhibitors was administrated. Diagnostic results: OCT - cystoid macular edema ; ERG: complete lack of response of rodes and cones ; perimetry: concentric narrowing of visual fields ; serology negative ; chest X-ray unremarkable ; extremely high concentration of plasma ornithine (1039) due to the lack of mitochondrial enzyme ornithine aminotransferase - gyrate atrophy of chorioretina was established. Molecular analisis revealed the homozygous mutation c.868_870delCTT (p.Leu290del) in exon 7 of OAT gene. The therapy with pyridoxine (vitamin B6), cofactor of ornithine aminotransferase, slightly reduced serum ornithine ; low-protein diet was introduced, but the response with ornithine level was insufficient. There was transient decrease of bilateral cystoid macular edema, but the progression of periferal atrophyc lesions continued. Therefore, treatment with amino acid L-lysine was introduced in the therapy with the aim to lower the reabsorption of ornithine and arginine in the kidneys. Conclusions: Although it seems that the posterior pole of the eye is preserved, cystoid macular edema in gyrate atrophy is a major cause of poor visual acuity. Despite pyridoxine treatment, low- protein diet and antiedematous therapy, macular edema persist and there is a notable progression of peripheral atrophic chorioretinal lesions. It is possible that L- lyosine supplementation would result in desired decrease of plasma ornithine concentration and subsequent clinical improvement.

U Odjelu za laboratorijsku dijagnostiku nasljednih metaboličkih bolesti Kliničkog bolničkog centra Zagreb već dugi niz godina sakupljaju se, izoliraju, analiziraju, skladište i distribuiraju različite vrste bioloških uzoraka (kožni fibroblasti, skeletni mioblasti i različiti humani bioptati pacijenata s rijetkim nasljednim metaboličkim bolestima). Stanične kulture fibroblasta i mioblasta daju mogućnosti proučavanja in vitro brojnih svojstava stanica vezivnog tkiva i mišićnih stanica što je izrazito važno za razjašnjavanje brojnih prirođenih i stečenih bolesti, za razumijevanje fizioloških zbivanja u organizmu kao i preduvjet za proučavanje brojnih lijekova. Biološki uzorci se skladište u različite biobanke i nephodni su za dijagnostiku i istraživanje u bilo kojem medicinskom području a posebno u području rijetkih bolesti. Na razini EU uočene su velike razlike u provedbi važećih direktiva, što predstavlja dodatni problem u iskoristivosti uzoraka pohranjenih u biobankama te posljedično umanjuje javnu zdravstvenu korist tih zbirki. Zato se u ovom diplomskom radu opisuje optimizacija metoda prikupljanja, uzgoja, skladištenja i raspodjele humanih fibroblasta, skeletnih mioblasta te bioptata mišića koji u Hrvatskoj otvaraju nove mogućnosti dijagnostike i istraživanja s krajnjim ciljem stvaranja preduvjeta za osnivanje biobanke tkiva i stanica za rijetke bolesti kao i usklađivanje biobanke s europskom biobankom. Different types of biological samples (skin fibroblasts and skeletal myoblasts, different human biopsy specimens of patients with rare inherited metabolic diseases) have been collected, isolated, analyzed, stored and distributed for many years in Clinical Unit for Hereditary Metabolic Diseases, Department of Laboratory Diagnostics, University Hospital Center Zagreb. Fibroblast and myoblast cell cultures provide the possibility of in vitro research of numerous properties of connective tissue and muscular cells, which is particularly important in elucidating many congenital and acquired diseases, for understanding of physiological processes in the body, and as a precondition for studying many medications. Biological samples are stored in different biobanks and are necessary for diagnostics and research in every medical field, particularly in the field of rare diseases. At EU level, considerable differences have been observed in implementation of applicable directives, which is an additional problem in utilization of samples stored in biobanks and which consequently reduces public health benefits from such sample collections. For this reason, this graduation thesis describes optimization of methods for collection, culture, storage, and distribution of human fibroblasts, skeletal myoblasts and muscle biopsy specimens which open in Croatia new possibilities for diagnostics and research with the ultimate goal of creating preconditions for founding a tissue and cell biobank for rare diseases and its harmonization with European biobank.

Background: S-adenosylhomocysteine hydrolase (SAHH)deficiency is an autosomal recessive methionine cycle disorder. Pathogenesis seems to be complex and inhibit ion of methyl t ransferases due to decreased Sadenosylmethionine/ S-adenosylhomocysteine (AdoMet/AdoHcy) ratio probably contributes to it. Biochemical hallmarks of the disease are elevated AdoHcy, AdoMet and hypermethioninemia. Clinical presentation is variable, but muscle disease with high CK is a constant feature. Methods:We performed pathological studies and muscle MRI to get insight into muscular pathology, and established myoblast cell cultures to test the effect of altered methylation index on muscle in SAHH deficient patients. Results: Muscle biopsy demonstrated destructive myopathy with myelin figures and immunohistochemistry revealed normal expression of muscle structural proteins.Muscle MRI showed degeneration predominantly affecting lower leg muscles and progression correlated with age. Muscle spectroscopy showed increased lipid and water peaks consistent with fatty degeneration and edema.Results on myoblast culture showed different methylation index and structural differences between SAHH deficient and control cell lines under various concentrations of AdoMet and AdoHcy. Conclusion: The pattern of affected muscles and progression with age indicate a contraction-induced injury as seen in dystrophinopathies. Altered methylation seems to be a contributing factor to the muscle pathology. Better understanding of pathogenesis could lead to a more tailored treatment.

Argininosukcinična acidurija (ASA) je autosomno recesivna aminoacidopatija prouzročena manjkom enzima iz ciklusa ureje argininosukcinat-liaze (ASL) koji cijepa argininosukciničnu kiselinu na arginin i fumarat. Bolest se očituje u dojenačkoj dobi s izrazitom hiperamonijemijom i kliničkim manifestacijama koje uključuju letargiju, somnolentnost, gubitak apetita, povraćanje, tahipneju, respiracijsku alkalozu i progresivnu encefalopatiju. Kako je hiperamonijemija stanje koje zahtijeva hitan terapijski pristup, za pravovremenu dijagnozu nužno je prepoznati manjkavi enzim iz ciklusa ureje mjerenjem koncentracija aminokiselina u plazmi i urinu. Muško novorođenče s hiperamonijemijom nepoznate etiologije. Aminokiseline su kvantificirane metodom tandemske spektrometrije masa udružene s tekućinskom kromatografijom visoke djelotvornosti (API 3200, tvrtka AB Sciex ; UPLC Nexera, tvrtka Shimadzu) uz primjenu reagensa aTRAQ™ tvrtke AB Sciex. Navedenom metodom moguće je istovremeno kvantificirati 45 aminokiselina u plazmi uz interne standarde poznatih koncentracija za svaku aminokiselinu. Analizirane su i sve kodirajuće regije gena za ASL u suradnom inozemnom centru University Children's Hospital u Zürichu, Švicarska. Rezultati Uz prisutnu izrazitu hiperamonijemiju (1047 µmol/L ; N 24-48 µM), u nalazu aminokiselina u plazmi nađene su izrazito povišene koncentracije citrulina (415 µM ; N 9- 35 µM) i argininosukcinične kiseline (1308 µM) koja se ne nalazi u zdravih ljudi. Koncentracija arginina bila je granično niska (18 µM ; N 6-130 µM). Na osnovu ovog nalaza postavljena je dijagnoza argininosukcinične acidurije. U skladu s postavljenom dijagnozom nađeno je da je pacijent homozigot za još neopisanu besmislenu (engl. non- sense) mutaciju c.1128C>A p.(Tyr376*) u genu za ASL. Mjerenje koncentracije aminokiselina metodom tandemske spektrometrije masa nužno je kod svih stanja s hiperamonijemijom nepoznatog uzroka, a osobito u diferencijalnoj dijagnostici poremećaja ciklusa ureje. Također je u dijagnosticiranih bolesnika nužno uz amonijak mjeriti i aminokiseline u plazmi i urinu kao jedan od parametara pri praćenju uspješnosti liječenja.

Background and objective: Citrullinemia type 1 (CTLN 1) is a rare autosomal recessive disorder caused by deficiency of argininosuccinate synthetase. CTLN1 presents as a clinical spectrum ranging from classic neonatal form to late-onset and even asymptomic form. Some women remain undiag-nosed until acute deteriora tion during pregnancy or post partum period. There are no clear recommendations for man- agement of newly diagnosed illness during pregnancy (pregnant women). Case report: A 20-year old women presented at 27th week of gestation with acute liver failure and encephalopathy. As plasma ammonia was high (220 μmol/L) metabolic work-up was performed. Plasma citrulline (921 μmol/L ; N 10-55 μmol/L) and glutamine (1141 μmol/L ; N 340-740 μmol/L) were high, arginine and argininosuccinate were unmeasurably low. Orotic acid excretion in urine was elevated. Treatment with sufficient caloric intake and low protein diet, arginine supplementation and sodium benzoate resulted in complete recovery.Patient tolerated 1.1g protein/kgBW, and probably could more. Plasma ammonia remained normal and amino acids in acceptable range. In 39 th week the patient delivered a healthy baby. Conclusion: Dietary management is an essential part of treatment of pregnant women with citrullinemia type I and contributes to good outcome both for the mother and baby.

A 20-year-old pregnant woman presented at 27th week of gestation with acute liver failure and hyperammonemia. Plasma amino acids were analyzed using high performance liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS). Plasma citrulline (921 µmol/L ; N 10-55 µmol/L) and glutamine (1141 µmol/L ; N 340-740 µmol/L) were extremely high and arginine was unmeasurably low. There was no argininosuccinate in plasma. Based on these findings, citrullinemia type 1 was diagnosed. Routine weekly monitoring for ammonia and amino acid concentration continued until post partum period. The patient delivered a healthy baby. Women with newly diagnosed citrullinemia type 1 can have normal pregnancy outcomes if metabolic crisis is treated promptly and ammonia and amino acid levels are monitored regularly during pregnancy.

Biološki uzorci i baza podataka u biobanci tkiva i stanica su jako bitni za razumijevanje i liječenje svih rijetkih bolesti uključujući i nasljedne metaboličke bolesti čiji pacijenti umiru u ranim godinama života. Biološke materijale poput urina, pune krvi, plazme, leukocita, DNA, stanične kulture kožnih fibroblasta, skeletnih mioblasta te bioptate jetre i mišića, trebalo bi skladištiti u biobanku s ciljem dijagnostike, terapije i istraživanja. Uskladišteni biološki materijal pacijenata s rijetkim metaboličkim bolestima omogućava istraživanja i bolje razumijevanje genetske podloge bolesti. Mnogi pacijenti s rijetkim bolestima imaju zajedničke simptome, ali za prepoznavanje vrste bolesti i pravovremenu terapiju neophodna je suradnja vrhunskih stručnjaka, od liječnika na odjelu do genetičara, biokemičara i molekularnih biologa u laboratoriju. Stanične kulture fibroblasta i mioblasta daju mogućnosti proučavanja in vitro brojnih svojstava stanica vezivnog tkiva i mišićnih stanica što je izrazito važno za razjašnjavanje brojnih prirođenih i stečenih bolesti, za razumijevanje fizioloških zbivanja u organizmu kao i preduvjet za proučavanje brojnih lijekova. Na razini EU uočene su velike razlike u provedbi važećih direktiva, što predstavlja dodatni problem u iskoristivosti uzoraka pohranjenih u biobankama te posljedično umanjuje javnu zdravstvenu korist tih zbirki. Biološki uzorci pacijenata kod kojih postoji sumnja na nasljednu metaboličku bolest prikupljeni su u skladu s hrvatskim i europskim zakonima. Komadić ljudske kože dostavlja se u mediju DMEM (engl. Dulbecco's modification of Eagle's medium) s dodatkom 20 % FBS (engl. Fetal bovine serum), 1% L-glutamina i 1% antibiotika (penicilin i streptomicin). Biopsija se usitni skalpelom na manje komadiće bez predhodnog odvajanja epidermisa od dermisa u kabinetu za sterilan rad. Medij treba mijenjati svaka 72 sata. Kada je primarna kultura dostigla konfluenciju oko 70%, stanice se tripsiniziraju (tripsin 0, 25 % i EDTA 0, 02 %), centrifugiraju (1100 rpm kroz 5 minuta) te se jedan dio zaledi, a drugi dio zasijava u nove subkulture. Uzorak skeletnog mišića prenosi se u prethodno pripremljene sterilne epruvete s proliferacijskim medijem. Osnovni medij je komercijalni medij Skeletal Muscle Cell Growht Medium tvrtke Promocell s dodatkom 5% faktora rasta. Dodatak čine epidermalni faktor rasta, faktor rasta fibroblasta i inzulin. U medij se dodaje 10% FBS, 1, 5% glutamina i antibiotika (penicilin i streptomicin). Svi postupci stanične kultivacije izvode se u sterilnim uvjetima s laminarnim protokom zraka. Tkivne matične stanice izoliraju se enzimskom digestijom u mediju koji sadrži tripsin iz bioptata skeletnog mišića bolesnika s nasljednom metaboličkom miopatijom. Ovako dobiveni mioblasti, uz manji dio fibroblasta, zasijavaju se u kultivacijske bočice u proliferacijskom mediju i pohranjuju u inkubator na +37 ˚C s 5% CO2. Za pročišćavanje mioblasta od fibroblasta koristi se magnetski MiniMACS-separator (engl. Magnetic-activated cell sorting). Mioblasti se obilježavaju specifičnim monoklonskim protutijelom (NCAM/CD56, Invitrogen) i IgG-mikrokuglicama, a zatim se čistoća provjerava na protočnom citometru. Pročišćeni mioblasti mogu se zamrznuti i dugotrajno čuvati u tekućem dušiku. Stanična kultura kožnih fibroblasta je optimalan a u većini slučajeva i neophodan biološki materijal za provođenje testova potvrde lizosomskih bolesti nakupljanja. Od iznimne je važnosti uspostava kulture mioblasta kao modela za daljnje istraživanje patogeneze miopatija i matičnih stanica. Nakon što su uspostavljene primarne i sekundarne kulture mioblasta te nakon što su mioblasti pročišćeni magnetskim separatorom, diferencirani su u miotube. Kontrola pročišćenosti mioblasta i vijabilnost stanica provjerene su metodom protočne citometrije i iznosila je 90%.Optimizirana je metoda prikupljanja, uzgoja, skladištenja i raspodjele humanih fibroblasta, skeletnih mioblasta koji u Hrvatskoj otvaraju nove mogućnosti dijagnostike i istraživanja na tkivnim matičnim stanicama s krajnjim ciljem stvaranja preduvjeta za osnivanje biobanke tkiva i stanica za rijetke bolesti kao i usklađivanje biobanke s europskom biobankom.

Objectives Rare diseases are a clinically heterogeneous group of disorders with prevalence less than 5/10000. Only 6-8% of the world’s population is likely to be affected by some form of rare disease. Since these diseases affect multiple organ systems and do not have characteristic symptoms, interdisciplinary cooperation is essential to establish diagnosis and include appropriate treatment. Life expectancy in these patients is often reduced ; however, if detected in time, many of these diseases can be successfully treated. Inherited metabolic diseases form a small group of rare diseases diagnosed not only using conventional biological material (blood, urine, cerebrospinal fluid) but also tissue biopsy specimens are often included in the diagnostic process. In the Clinical Unit for Hereditary Metabolic Diseases of the University Hospital Centre Zagreb, different types of progenitor cells (dermal fibroblasts and skeletal myoblasts) and various human biopsy specimens (muscle, liver) of patients with rare hereditary metabolic diseases have been collected, processed, stored and handled for many years. Until now, Croatia had no organized biobank that would meet common European standards and is currently in process of establishing the first non-profit tissue and cell biobank of patients with inborn errors of metabolism. The main goal is to optimize the establishment of cell cultures and ultimately implement and harmonize Standard Operating Procedures in accordance with the European Network of Biobanks (EuroBioBanks, EBB) and ISO 15189 standards. Materials and Methods Progenitor cells were isolated from skin and skeletal muscle biopsy specimens of patients with inborn errors of metabolism. After the establishment of primary and secondary cultures, the cells were frozen in liquid nitrogen. Dulbecco's modification of Eagle's medium supplemented with 20% of fetal bovine serum (FBS), 1% of L-glutamine and 1% of antibiotics was used in the cultivation of fibroblasts. Myoblasts, with a small number of fibroblasts, were cultured in a Skeletal Muscle Growth Medium, SGM (PromoCell, Germany) supplemented with 5% of growth factors (epidermal growth factor, fibroblast growth factor and insulin). Into the medium 10% of FBS, 1.5% of glutamine and antibiotics were added. Purification of myoblasts from fibroblasts was performed using Magnetic- Activated Cell Sorting Technology (Miltenyi Biotec, Germany). IgG-microbeads coated in specific primary antibody NCAM/CD56 were used to label myoblasts and separate them from unlabelled cells. Purity of myoblasts obtained by immunomagnetic sorting was assessed using flow cytometer before the storage in liquid nitrogen. Results The number of established cell cultures (1002 human fibroblast cultures and 48 skeletal myoblast cultures) together with the number of human biopsy specimens (337) isolated from patients with rare hereditary diseases was entered in the first Croatian biobank database. The purity of myoblast cell cultures was nearly 90%. The entire process of collecting, processing, storage and handling of samples was performed in concordance with valid European standards. Conclusion The first step in establishment of Croatian tissue and cell biobank has been taken. Requirements for data networking and harmonization of Croatian biobank according to the EBB and ISO 15189 standards have been accomplished. Cell culture of human myoblasts opens up new possibilities in the field of research of adult stem cells.

U proteklih 10-15 godina u znatnom je porastu rutinska primjena sustava tekućinske kromatografije visoke djelotvornosti vezane s tandemskom spektrometrijom masa (engl. High performance liquid chromatography coupled with mass spectrometry ; LC-MS/MS) u kliničkim laboratorijima. Ova tehnologija omogućuje kvalitativno i kvantitativno mjerenje mnogih analita iz različitih uzoraka biološkog materijala. Pri razvoju metoda optimiraju se parametri pripreme uzoraka, kromatografski uvjeti te uvjeti na spektrometru masa. Tekućinskom kromatografijom visoke djelotvornosti (LC) analiti od interesa razdvajaju se na koloni, u spektrometru masa (MS/MS) se ioniziraju, dodatno odabiru te na kraju detektiraju na temelju odnosa mase i naboja. Za većinu metoda nužno je rabiti obje komponenete sustava LC-MS/MS. Metabolički laboratoriji koji na raspolaganju imaju više LC- MS/MS sustava različitih osjetljivosti mogu analizirati: aktivnosti lizosomskih enzima, biomarkere za praćenje tijeka liječenja lizosomskih bolesti nakupljanja, karnitin, metilmalonsku kiselinu, oligosaharide, glikozaminoglikane, kreatin, homocistein, vitamine, neuroprijenosnike, purine i pirimidine i dr. Nakon konzultacije s kliničarima odredili smo prioritete na našem LC-MS/MS sustavu (Shimadzu Nexera UPLC - AB Sciex API 3200TM): - Analiza profila aminokiselina i acil-karnitina iz suhe kapi krvi na filtar-papiru u svrhu provođenja proširenoga novorođenačkog probira za nasljedne metaboličke bolesti. Do sada je napravljena skraćena validacija metode i rezultati su prihvatljivi. - Aminokiseline u plazmi, likvoru i urinu u svrhu dijagnostike i praćenja tijeka liječenja nasljednih metaboličkih poremećaja. Metoda je validirana i u rutinskoj je primjeni. - Profil acilkarnitina iz plazme u svrhu dijagnostike poremećaja beta-oksidacije masnih kiselina. - S-adenozilmetionin i S- adenozilhomocistein u plazmi u svrhu dijagnostike poremećaja metilacijskog ciklusa. - Masne kiseline vrlo dugih lanaca u plazmi u svrhu dijagnostike peroksisomskih bolesti. LC-MS/MS je tehnologija koja će postupno zauzimati sve važnije mjesto u kliničkim laboratorijima, a posebice u dijagnostici nasljednih metaboličkih bolesti. Njezine su glavne prednosti znatno manja potrebna količina uzorka, visoka osjetljivost i specifičnost te kratko vrijeme izvođenja analiza. Ipak treba istaknuti da zbog svoje složenosti iziskuje stručno educirano osoblje koje će se moći nositi sa svim izazovima ove višenamjenske tehnologije.

Introduction High performance liquid chromatography, combined with tandem mass spectrometry (LC-MS/MS), is a technology used for quantitative measurement of of numerous metabolites, including amino acids in plasma, urine and cerebrospinal fluid. For analysis, only 40 µL of sample is needed to quantify 45 amino acids in a very short time span. Aim Shortened validation procedure for a method of quantifying 45 amino acids in plasma. Materials and methods The validation was carried out using aTRAQ™ reagent manufactured by AB Sciex. Amino acids were chromatographically separated on the AB Sciex C18 column (5 µm, 4.6 mm x 150 mm) on UPLC-NEXERA instrument manufactured by Shimadzu, with binary gradient of water (mobile phase A) and methanol (mobile phase B) and addition of 0.1% of formic acid and 0.01% of heptafluorobutyric acid to both mobile phases. Amino acids were quantified on API 3200 mass spectrometer manufactured by AB Sciex. The following commercial standards were used for validation: AMI-01 produced by SKML (MCA Laboratory, the Netherlands), preparations of A6407 and A6282 amino acid standards produced by Sigma, and laboratory preparation of glutamine standard. Shortened validation procedure for the method included testing of between-run and within- run imprecision, inaccuracy, limit of detection, limit of quantitation and linearity. Results Calculated values of the coefficient of variation (CV) for within-run and between-run imprecision and inaccuracy for most amino acids were lower than 20%, with the exception of glutamine (CV=24%). Limit of detection (LoD) and limit of quantitation (LoQ) were lower than 2 µmol/L for most amino acids except for serine, glycine and ß- alanine which had somewhat higher LoQ. The method was linear up to 1000 µmol/L for most amino acids. Conclusion Based on results of shortened validation procedure, it was concluded that the method is acceptable for routine laboratory practice. Advantages of the method are significantly low sample volume needed and reduced analysis time

UVOD Manjak S-adenozilhomocistein hidrolaze (SAHH) dokazan je dosad u devet bolesnika. Iako je klinička slika varijabilna, svi bolesnici koji boluju od ove multisistemske bolesti od rođenja imaju miopatiju i značajno povišenu serumsku aktivnost enzima kreatin kinaze. Za ovu bolest su specifični izrazito povišena serumska koncentracija S-adenozilmetionina i posebno S- adenozilhomocisteina te snižen omjer S- adenozilmetionina/S-adenozilhomocisteina (SAM/SAH) (metilacijski indeks). Za sada dostupno liječenje (dijeta s ograničenim unosom metionina, dodatni unos kreatina, cisteina i fosfatidilkolina) može poboljšati stanje bolesnika, posebice ako se rano započne s liječenjem. Liječenje djeluje na mnoge elemente bolesti, ali u mišićima izostaje repozitivan učinak ili je on mnogo manji. U skladu s tim poboljšavaju se i laboratorijski pokazatelji osim aktivnosti kreatin kinaze (CK-MM, mišićni oblik) koja ostaje značajno povišena. Svjetlosnim mikroskopom vidljivi su znakovi destruktivne miopatije, ali ne u svih bolesnika jednako. CILJ Uspostava kulture mioblasta kao modela koji će omogućiti daljnje proučavanje patogeneze manjka SAHH na razini metabolita, proteina i gena. MATERIJALI I METODE Bioptat skeletnog mišića veličine 1 mm3, usitnjen je skalpelom, u kabinetu za sterilan rad. Uz pomoć enzimatske digestije, izolirani su mioblasti. Stanice su uzgajane u CO2 inkubatoru na 37˚C, u mediju s faktorima rasta (Skeletal Muscle Growth Media) u sterilnim kultivacijskim bočicama 25/75 cm2. Nakon uspostave primarne kulture, medij je mjenjan svaka tri dana. Nakon što su mioblasti postigli konfluenciju 70- 80%, stanice su tripsinizacijom podjeljene u subkulture. REZULTATI Uspostavljena je primarna kultura mioblasta i fibroblasta.Uzgojene su subkulture mioblasta i fibroblasta. Odvojeni su mioblasti od fibroblasta korištenjem magnetskog staničnog separatora (Miltenyi Biotec) i antitijela NCAM i Mouse IgG1. Optimiziran je broj pročišćenih mioblasta u kultivacijskim bočicama. Diferencirani su mioblasti u miotube. Napravljena je baza mioblasta od ranih do kasnih pasaža. Stanice su uskladištene u tekući dušik, oko 1, 5 x 106 mioblasta po ampuli. Provjerena je vijabilnost mioblasta nakon zaleđivanja (oko 80%). Metodom protočne citometrije dokazana je čistoća mioblasta 81, 4% (CD56+/CD45-). ZAKLJUČAK Mogućnost uspostave i pohrane staničnih kultura mioblasta bolesnika s manjkom SAHH preduvjet je za daljnje proučavanje ove kongenitalne miopatije. To je ujedno i put koji otvara mogućnosti za otkrivanje uzroka propadanja mišića i u drugim patološkim stanjima sa smanjenim metilacijskim indeksom i/ili poremećenom metilacijom. Kultura mioblasta otvara nove mogućnosti u području rijetkih bolesti, prvenstveno miopatija. Takav biološki materijal omogućuje studije staničnih i molekularnih mehanizama koje mogu doprinijeti razumijevanju patofiziologije, otkrivanju novih biomarkera i ubrzanju razvoja novih terapijskih pristupa.

U rastućem broju nasljednih metaboličkih bolesti lizosomske bolesti nakupljanja čine relativno malu skupinu od 60-tak bolesti. Njihova podskupina, mukopolisaharidoze (MPS), obuhvaća samo sedam tipova bolesti (uz podtipove), koje su zbog patofi ziološke kompleksnosti, progresivnog tijeka, multiorganske zahvaćenosti te izrazite kliničke heterogenosti i danas izazov suvremenoj medicini. U posljednjih desetak godina velik je broj bolesnika s mukopolisaharidozama uključen u različite programe liječenja. U tijeku je niz kliničkih ispitivanja kao i najave novih mogućih pristupa liječenju i specifi čnijih biomarkera. Potreba za što ranijim postavljanjem dijagnoze i poduzimanjem pravodobnog liječenja dovela je do znatno jednostavnijeg i bržeg slanja uzoraka u laboratorije. Sve češće fi ltar-papir zamjenjuje epruvetu, a dijagnostičke se metode prilagođavaju tehnologiji tandemske spektrometrije masa (MS/MS). Sve ove činjenice upućuju na to da ovoj podskupini lizosomskih bolesti treba posvetiti dodatnu pozornost.

Uvod: Prošireni novorođenački probir mjerenjem aminokiselina i acil-karnitina tandemskom spektrometrijom masa (LC-MS/MS) omogućuje pravovremeno otkrivanje i odgovarajuće liječenje većeg broja nasljednih metaboličkih bolesti. Cilj: Skraćena validacija semikvantitativne metode za 14 aminokiselina i 14 acil-karnitina iz uzorka suhe kapi krvi na filtar papiru.Materijali i metode: Reagens kit uz derivatizaciju MassChrom® tvrtke Chromsystems. LC-MS/MS: UPLC-NEXERA, Shimadzu - API 3200, ABSciex. Validacija metode uključivala je ispitivanje nepreciznosti u seriji, nepreciznosti iz dana u dan i netočnosti u kontrolnim komercijalnim uzorcima niske i visoke koncentracije MassCheck® tvrtke Chromsystems.Rezultati: Izračunate vrijednosti koeficijenta varijacije (CV) za nepreciznost u seriji i nepreciznosti iz dana u dan za većinu aminokiselina i acil-karnitina su

Background: Systemic control of entire laboratory process and monitoring and management of nonconformities are mandatory procedures for every medical laboratory with established quality control system. Along with the most common nonconformities related to sample quality (hemolysis, clot...), a significant number of the nonconformity entitled “sample not delivered” was recorded at Department of Laboratory Diagnostics in 2011. The number of such cases was considerably reduced after introduction of Sample Reader inSIGHT2 device for automated reading of barcoded samples. Materials and methods: Prior to introduction of Sample Reader inSIGHT2 device, there was no possibility for individual receipt of requests for laboratory management in the laboratory information system (LIS), regardless of whether all samples associated with an individual request have been delivered to the laboratory. The Sample reader inSIGHT2 accepts requests only for the samples delivered to the laboratory and barcoded with the label generated from the hospital information system (HIS) or LIS. Communication with the user takes place via a touch sensitive screen. The device informs the user if a sample has already been received and recorded in LIS and if it is acceptable with regard to the sampling time. The files with accreditation-related data (sample receipt time, identification of the user who has received a sample, identification numbers of received samples) are kept for each received sample while, for the purpose of subsequent forensic management, each sample or test-tube rack is photographed. Results: After application of Sample Reader inSIGHT2, the nonconformity “sample not delivered” was recorded in 154 cases, as compared to 1092 cases in 2011 (for the periods of three months), while the average receipt time has been reduced significantly. Conclusion: The application of the device for automated reading of barcoded samples at sample receipt unit of the Department of Laboratory Diagnostics, has allowed rapid and accurate reading of barcoded samples and also a reduced number of the cases of nonconformity entitled “sample not delivered”.

TRMU gene mutations cause a deficiency of an enzyme required for the 2-thio modification of mitochondrial tRNA and lead to a combined respiratory chain deficiency secondary to a defect in mitochondrial DNA translation. Clinically they present with acute liver failure in early infancy. Patients who survive the acute phase of disease recover liver function and do not experience recurrence of disease. Various other organ involvement have been described indicating various phenotypes of the disease. It seems that there is no genotype-phenotype correlation. We report an infant with reversible liver failure caused by novel TRMU gene mutation who seems to had among worst biochemical findings among all survivors so far. In the acute phase of disease even liver transplantation was considered. Our patient recovered after a couple of months of intensive supportive care. This case points to necessity to consider TRMU gene mutations in infants with acute liver failure as it is probably much more common than reported and to be cautious concerning liver transplantation because this can turn out to be unnecessary high risk procedure. Recognizing patients with TRMU deficiency and identifying factors which influence the prognosis is crucial for optimal management of these patients.

Introduction Skeletal muscle is composed of muscle cells (myoblasts) which unite into myofibrils and later in the muscle fibers. Activation of myoblasts after muscle injury or muscle disease is an important step in the process of muscle regeneration. During muscle regeneration myoblasts enter the proliferative process at the end of which the majority of myoblasts are differentiated in myotube. Materials and Methods Myoblasts were isolated by enzymatic digestion of skeletal muscle biopsy. After the cells reached a density about 70-80%, muscle stem cells were prepared for the separation of myoblasts from fibroblasts. Myoblasts were first characterized by specific monoclonal antibody (NCAM/CD56) and IgG microspheres boding. Then the Magnetic-activated cell sorting (Miltenyl Biotec, GmbH) separates from fibroblasts. Differentiation of myoblasts in the myotubes can be traced by the phase-contrast microscope. Results We have established a cultures of stem cells, myoblasts and fibroblasts from a very small piece of muscle biopsy. Using a magnetic cell separator, myoblasts were separated from fibroblasts and as such stored in liquid nitrogen. Purity of myoblasts was confirmed with markers CD 56+ and CD 45- by flow cytometry. It was confirmed that 81.4% of the cells carry a myoblast marker. Conclusion Cell culture of human myoblasts opens up new possibilities in the field of research of adult stem cells and rare diseases, especially mitochondrial myopathy. This biological material allows the study of cellular and molecular mechanisms that may contribute to the understanding of the pathophysiology and new therapeutic approaches by using stem cells.

Background: On average, about 200 outpatients are admitted daily at the Department of Laboratory Diagnostics (DOLD), University Hospital Center Zagreb. Relocation of the Clinical Unit for Patient Admission and Management to new premises has entailed reorganization of outpatient admission and sampling of biological material. HAVIS, an audio-visual system for calling out patients has been integrated with the currently used hospital (HIS) and laboratory information system (LIS), while blood sampling test-tubes are barcode labelled in advance. Materials and methods: Upon arrival to laboratory reception desk, patients present the referral for laboratory tests and are given a number generated by the HAVIS system. A copy of the number is enclosed with the referral, and basic patient data and laboratory tests are entered into HIS. All test requests are transferred to LIS using the HL7 communication protocol. Phlebotomy is performed in five separate rooms in order to ensure patient privacy. A computer is in each of the rooms, with all three applications (HIS, LIS and HAVIS). A patient is referred to the room for venipunction through the HAVIS system, i.e. digital voice and visual information on a display in the laboratory waiting room. After checking patient’s identity, laboratory staff performs blood sampling. Information on laboratory tests for a selected patient is displayed on computer screen at the LIS interface entitled: “Entered requests”, and data on a number and type of previously labelled test- tubes are added. After sampling, samples are transported in closed containers to the worksite. Results: In the first half of year 2012, 22, 926 outpatients were managed at DOLD. Average time from presentation of referral to patient’s entry into phlebotomy room was 0:35:18 minutes (min: 0:14:14, max: 0:58:33), depending on the type and complexity of referral. Application of test-tubes labelled in advance reduced patient management time and ensured privacy (patient’s name and surname are not written on the test-tube). Also, expansion of the existing LIS with a module that, depending on the number of requested tests, determines the minimum number of necessary blood test-tubes, helped reduce the number of unnecessarily sampled test-tubes. Conclusion: Better work organization and implementation of information systems improved the entire process of laboratory management of outpatients and thus contributed to increased quality of patient health care.

U radu će biti istražena genotipizacija gena CFTR, pronalazak novih mutacija, učestalost najčešće mutacije u Hrvatskoj u odnosu na podatke u susjednoj regiji i u svijetu. Koristiti će se umnažanje ulomka DNA metodom PCR (engl. polymerase chain reaction) te fragmentarna analiza kapilarnom elektroforezom.

Background: Cystic fibrosis (CF) is the most common autosomal recessive hereditary disease in Caucasians with an average incidence of 1:2500 newborns. CFTR gene codes for CFTR protein (cystic fibrosis transmembrane conductance regulator) which regulates the transport of chloride ions through cell membranes. Materials and methods: Genomic DNA of 65 CF patients was screened for 32 mutations by Cystic Fibrosis Genotyping Assay CFv3 (Abbott). Twelve patients who were found to be heterozygous for one CF mutation were further analysed by sequencing method for specific exons (7, 10, 11, 12 and 19) on AB Genetic Analyzer 3130XL, using BigDye Terminator Cycle Sequencing Kit v3.1 (Applied Biosystems). Results: Since year 2001, out of 65 patients detected to have CF, 42 were homozygous with the deltaF508 mutation and 11 were compound heterozygous. By using commercial kit we revealed 14 different mutations in patients with CF. Through sequencing exons 7, 10, 11, 12 and 19 in 12 heterozygous patients so far we have found five polymorphisms: M470V (c.1408A>G), 1898+152T/A (c.1766+152T>A), 3601-65C/A (c.3469- 65C>A), 1525- 61A/G (c.1393-61A>G) and R75Q (c.224G>A). These DNA polymorphisms will certainly contribute to understanding the sequential variants and clinical correlation. Other CFTR exons still remain to be analyzed. Conclusions: This paper investigates variants in CFTR gene other than the panel covered by commercial kit. By collecting information about similar cases and studying them, we are likely to notice a link between new mutations, polymorphisms and clinical features.

S-adenosylhomocysteine hydrolase deficiency (SAHH) is a rare disease, described for the first time in 2004. SAHH is one of the enzymes in methionine cycle, in which it catalyzes the hydrolysis of S- adenosyhomocysteine (AdoHcy) to adenosine (Ado) and homocysteine (Hcy). AdoHcy is formed by transmethylation processes in which CH3 group is transfered from S-adenosylmethionine (AdoMet) to numerous methyl acceptor molecules using methyltransferases (MT). Ratio of AdoMet/AdoHcy concentration is called methylation index of the cell. Methylation index is considered to be one of the key players in disease development by inhibiting metyltransferases and leading to misregulated methylation of molecules within cell. The purpose of this work is to determine methylation index in fibroblast cell culture with S-adenosylhomocysteine hydrolase deficiency. Fibroblasts cultivated in 75 cm2 flasks were washed in phosphate buffer solution. Immediately after washing, 5 ml of 0.6 N perchloric acid with N-6-methyladenosine as an internal standard was added to cells. Cell debris was collected into tubes by scraping. The tubes were shaken thoroughly, placed on ice and centrifuged (4 000 x g, 10 min at +4 °C). The supernatant was adjusted to pH 5.5 by adding 2 M K2CO3/1 M KH2PO4 and then centrifuged. Supernatant was applied onto solid- phase extraction column (BondElut LRC-PBA). Elution of AdoHcy, AdoMet and internal standard was performed by using 0.1 M HCl and the eluate was analysed by high performance liquid chromatography (MN CC 250/4.6 NUCLEODUR C18 Gravity 5 m) with UV-detection (258 nm). The mobile phase consisted of solvent A (50 mM natrium dihydrogenphosphate, 8 mM heptanesulfonic acid sodium salt with addition of o-phosphoric acid, pH 3, 0) and solvent B (methanol). High performance liquid chromatography was previously used for AdoMet/AdoHcy ratio analysis in whole blood. Here, we demonstrate that AdoMet/AdoHcy ratio can be measured not only in whole blood, but also in cells, which could be useful biomarker at cellular level for the diagnosis of S-adenosylhomocysteine hydrolase deficiency. Our results suggest that the AdoMet/AdoHcy ratio is significantly lower in SAHH deficient fibroblast cells when compared to control.

The Clinical Institute of Laboratory Diagnosis is in the process of preparing for accreditation according to the internationally accepted ISO 15 189. In this complex and challenging process of implementation, quality indicators and eligibility criteria are defined. Although the number of analysis from year to year is growing, our policy is to reduce the number of non compliance to a minimum by identifying, recording, resolution and analysis of non compliance in all aspects of testing with special emphasis on preanalytical phase. Program writing and recording nonconformities issue findings within a specific time and statistical data processing were performed using a computer program support in Laboratory informatic system (LIS). Indicators quality and eligibility criteria are defined by incidence of hemolysis in the serum/plasma (90%). In the last quarter of 2010. from the number of 83 018 patients and 159 144 number of samples submitted, there were 1712 nonconformities, which is 0.01% of total samples. The largest number of nonconformities do not passed urine 0.58%, and the number of samples hemolysis 0.14%, sample for hematological tests is not passed 0.09%. In the annual report in the first two quarters of 2010. year nonconformities were recorded manually, there we observed large discrepancies. Last quarter record an increase of about 80% of nonconforming due to the introduced electronic recording of the findings. The introduction of electronic recording of nonconformities in the last quarter of 2010. year there was a significant increase in the number of nonconformities recording which is much more realistic.

Introduction: Increasing evidence suggest that an interplay between T cells and fibroblasts plays a pivotal role in promoting matrix accumulation in systemic sclerosis (SSc). Aim: We investigated if the co-culturing of fibroblasts derived from SSc patients with autologous PBMCs could induce peculiar modifications in any cell types. Materials and methods: Fibroblasts were obtained from the skin of patients undergoing biopsy for diagnostic purposes. PBMCs were obtained from the same patients. All the patients were fully informed of the meaning of the study and accepted to donate blood. Cells were taken in culture at 37 ˚C5% CO2 for ten days, then cells were stained with monoclonal antibodies for HLA-DR, CD3, CD4, CD56, CD95, TCRab, TCRcd, After the staining, cells were analyzed by flow-cytometry using a FACScalibur. Results: We found that T cells bearing an ab receptor were expanded in the co-cultures. Moreover, these cells were positive for the expression of HLA-DR, suggesting an activation of T cells induced by co-culturing with autologous fibroblasts. No expansion of cd T cells nor CD56 positive T cells was found. SSc fibroblasts, which have already been reported to have a basal high expression of CD95 (FAS), were found to up-regulate FAS in these experiments. Conclusions: We recently showed that peripheral blood T cells from SSc patients expand when co-cultured with autologous fibroblasts and acquire the same T cell clonotypes that are increased in the affected skin. The results presented here further support that such expansion may be due to a specific antigenic activity of fibroblasts on T cells. In addition, the up-regulation of FAS expression on SSc fibroblasts may be related to phenotypic changes that indicate an increased ability of these cells to escape normal pathways leading to cell death. The meaning of these findings in the pathogenesis of SSc remains to be further investigated.

Opisujemo naša rezultate u liječenju trinaestogodišnjeg dječaka koji je zadobio opsežne i duboke opekline strujom visokog napona koje su zahvaćale 75% tijela. Tad smo prvi put primijenili novu metodu za tretman teških opeklina i kroničnih rana, koristeći suspenziju autolognih keratinocita u fibrinskom ljepilu, engl. Keratinocytes -Fibrin-Glue- Suspension (KFGS). Autologni epidermni presadci uzgojeni in vitro uspješno su primijenjeni u praksi, te potvrđuju mogućnost da će se djeca s opsežnim i dubokim opeklinama u Hrvatskoj moći liječiti i ovom naprednom i učinkovitom metodom.