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As a precious biological substance, donated blood has to be used in a sustainable, safe and efficient manner. Therefore good transfusion practice in modern surgery, anaesthesiology and intensive care is of key importance. New therapeutic methods, operations, transplantation of organs and hematopoietic stem cells often require intensive and long term supportive therapy including transfusions of blood components. Nowadays, transfusion service is confronted with continuously increasing demand for sufficient quantity of safe high quality blood, and on the other hand with limited resources of blood. The article deals with the current modern approaches for optimization of transfusion practice and Patient Blood Management that have to be implemented in Slovenian hospitals.

Extracellular membrane vesicles are fragments shed from plasma membranes off all cell types that are undergoing apoptosis or are being subjected to various types of stimulation or stress. Even in the process of programmed cell death (apoptosis), cell fall apart of varying size vesicles. They expose phosphatidylserine (PS) on the outer leaflet of their membrane, and bear surface membrane antigens reflecting their cellular origin. Extracellular membrane vesicles have been isolated from many types of biological fluids, including serum, cerebrospinal fluid, urine, saliva, tears and conditioned culture medium. Flow cytometry is one of the many different methodological approaches that have been used to analyze EMVs. The method attempts to characterize the EMVs cellular origin, size, population, number, and structure. EMVs are present and accumulate in blood products (erythrocytes, platelets) as well as in fresh frozen plasma during storage. The aim of this review is to highlight the importance of extracellular vesicles as a cell-to-cell communication system and the role in the pathogenesis of different diseases. Special emphasis will be given to the implication of extracellular membrane vesicles in blood products and their clinical relevance. Although our understanding of the role of EMVs in disease is far from comprehensive, they display promise as biomarkers for different diseases in the future and also as a marker of quality and safety in the quality control of blood products.

Gene therapy is the treatment of genetic disor - ders by introducing genetic material into cells. For this purpose, viral vectors and non-viral methods for introducing genes into cells are used, the later being in general less effective. After the enthusiastic beginings in 1990, gene ther - apy came to a standstill due to side effects of ad - enoviral vectors during gene therapy and when several patients developed leukemia. Nowadays, many studies are testing clinical efficacy of gene therapy to cure cancer, monogenetic disorders and cardiovascular deseases. In last few years we witnessed successful treatments of some patients with severe immune deficiency, Leber's congeni - tal amaurosis, thalassaemia and adrenoleuko - dystrophy. Recently, approval of the first gene therapy by European authorities was reported. It seems that gene therapy, regardless of the rela - tive delay in the 90's, is coming into forefront of advanced therapies.

Heparin-induced thrombocytopenia (HIT) is an immune-mediated reaction occurring in 3 - 5 % of patients receiving heparin therapy for more than 5 days, which can lead to arterial and venous thrombosis. It is caused by IgG antibodies directed against platelet factor 4/heparin complexes, leading to platelet activation, platelet depletion and thrombocytopenia. Prolonged therapy with heparin can result in serious adverse consequences, so prompt diagnosis is of key importance for the introduction of supplemental therapy. The diagnosis of HIT is based on clinical criteria as well as on serological and functional laboratory assays. Serological assays that identify the presence of platelet factor 4/heparin antibodies in the serum are not sufficient for confirmation of diagnosis because all detected antibodies are not capable of causing thrombocytopenia and thrombosis. Since the usual enzyme-immune assays detect the presence of antibodies but not also their pathogeneity it is recommended to confirm HIT with a functional assay that confirms the capacity of HIT antibodies to activate or aggregate platelets. One of newer functional methods is the flow cytometric analysis of P-selectin (CD62P) expression on the standard platelets after exposure to the patientćs serum. In our experience, this test, in parallele with standard serological tests, proved its reliability for the confirmation of clinical HIT diagnosis.

According to their capabilities for differentiation stem cells can be totipotent, pluripotent, multipotent and unipotent. After fertilization, zygote is the ultimate totipotent cell. Cells arising durring embryonic development have a lesser potency. Different pluripotent cell types are known: embryonic stem cells, embryonic germ cells, induced pluripotent stem cells, epiblast stem cells, and still controversial pluripotent stem cells isolated from the adult tissues. It is currently recognized that every tissue contains a small population of stem cells that are capable of self-renewal and differentiation. Embryonic stem cells have the broadest potential for differentiation because they are capable of turning into every cell type in our bodies. This is the reason why they are the most interesting for use in medicine. Lately, a lot of new studies have shown that somatic stem cells have broader potency than it has been expected until now and at the same time do not have any of embryonic stem cell drawbacks.

Umbilical cord blood (UCB) is an abundant source of haematopoietic stem cells (HSC) for allogeneic HSC transplant. In addition to HSCs, the cord blood and placenta contain ample nonhaematopoietic stem cells, which increasingly raise the interest of regenerative medicine. Public UCB banks have more than 500.000UCB units in international UCB registries that are available for the international exchange. It is estimated that additional two-fold number of units are held in the private UCB banks that store the cells for autologous purposes. Although the development of hybrid UCB banks was predicted in which the private units of UCB could be used for allogeneic purposes if needed, thishas not yet happened. The activity of UCB registries is complementary to the bone marrow donor registries, which keep the data of more than 16 million donors. The key data for international exchange of UCB are the HLA typing results and the stem cell count in the UCB units. The majority of international associations and bodies do not support the autologous UCB banking based on the current poor evidence on the usefulness of autologous UCBcells. However, this opinion is changing owing to the development of regenerative medicine so changes of the ethical and expert opinions are expected. In Slovenia, the only public UCB bank is maintained at the Blood Transfusion Centre of Slovenia, whereas the only private UCB bank is Biobanka.Since unlimited expansion in the number of UCB units does not increase the like-lihood of finding HLA-identical grafts, in Slovenia we plan to collect maximum up to 2000 UCB units. In order to foster the solidarity andequal accessibility, one needs to assure the use of UCB derived cells for children and adults for their possible treatment in the future.

Despite the progress in medicine, we are nowadays still faced with diseases and injuries, where with the knowledge, drugs and support technology availableto us, we cannot achieve the desired quality of patientćs life. Progress in stem cell biology may be the hope for certain diseases in the future. The purpose of this article is to present a rapidly advancing field ofstem cells. We summarize the properties of stem cells, various types of stemcells according to their origin from the embryonic or adult tissues, and according to their differentiation potential. Although numerous factors that determine the properties of stem cells are currently already known, we still need to know more about their mutual impact. We present the fields of clinicalmedicine where stem cells are already used for treatment, as well as those, for which there is, a justified hope that stem cell therapy will becomepart of routine treatment in the near future. The greatest potential of stem cells is probably their use in the treatment of degenerative diseases andtraumatic injuries, where we expect a major impact on the patientćs qualityof life. Similarly, the knowledge of stem cells has also contributed tounderstanding cancer diseases and thus open up opportunities for new approach to treating these diseases.

Background By introducing telemedicine, we would like to provide support for anew organization of the Slovenian transfusion service to ensure a uniform quality of services throughout the country. We would like to increase safety and to enable professional consultations by means of modern technology during periods when no transfusion medicine specialist is present at the transfusion departments. Methods A system that enables teleconsultations between individual transfusion institutions requires a safe and coded network, equipment for the capturing and processing of multimedia data, a videoconferencing system, and links to individual professional environments. An apparatus (Gelscope) has been developed to capture and process digitized images of laboratory data obtained by testing according to the gel method. Teleconsultations are performed as special scenarios which essentially includes questions passed by the on-duty doctor and qualified replies of the transfusion medicine specialist that are sent from the consultation center. Results An effective transfusion service requires the provision of uninterrupted on-duty coverage (24 hours/7 days) for each transfusion institution. Through the use of a teleconsultation system, it is possible to reduce the number of on-duty transfusion specialists from 11 to only 2. Furthermore, the system ensures a uniform quality of transfusion services overthe entire country. Validation of the pilot system has been completed and its results have shown that a transfusionćs services provided on a remote basis are equivalent in quality. Conclusions The teleconsultation system enables professional consultations via the use of modern technology. The implementation of this system will be continued and expanded from the current 4 system in use to 11 throughout Slovenia. The computer application is modularand it should also be suitable for use in other areas of medicine, suchas emergency medicine, etc.

Background. Even though blood transfusion is a relatively safe form of therapy, because of the eventual administrative errors in the transfusion chain between the blood donor and the recipient of blood, transfusion errors still occur. Therefore, it is imperative to ensure an utmost extent of safety and reliability of all transfusion related procedures. The safety of blood transfusion can be improved by preventive actions, i.e. implementation of the total quality management concept, haemovigilance and virtual transfusion laboratory. In the resulting system, the information web, robotics, computer sciences and communication technologies ensuresafe and reliable identificationof the patients, blood donors, corresponding test samples and blood products. Apart form this; the modern technologies enable the automationof laboratory testing, the integrity of laboratory results and enable an optimal use of blood. Conclusions. For an improved transfusion safety in Slovenia, adoption of corresponding prevention as well as haemovigilance is necessary. Identification errors can be prevented by implementation of the wrist bands systems with the code bars for the tagging of the patient and his biological samples, whereas the administrative errors in the blood bank and transfusion laboratory can be prevented by implementation of information systems and automation. We assume that the virtual transfusion laboratory will become an integral part of the new Slovenian transfusion web and will speed up, unify and simplify today's methods of ordering and administering blood products. (Abstract truncated at 2000 characters). Izhodišča. Kljub temu da je transfuzija relativno varna oblika zdravljenja, zaradi možnih administrativnih napak v transfuzijski verigi med krvodajalcem in bolnikom še vedno lahko prihaja do napak. Zato je treba zagotoviti čim večjo varnost in zanesljivost vseh postopkov v zvezi s transfuzijo krvi. Varnost transfuzije lahko povečamo predvsem s preventivnimi ukrepi, to je z uvedbo celostnega sistema kakovosti, sistema hemovigilance in z virtualnim transfuzijskim laboratorijem. V takem sistemu informacijska mreža, robotika, računalništvo in komunikacijske tehnologije zagotovijo zanesljivost identifikacije bolnika-prejemnika krvi, dajalca krvi, ustreznih vzorcev krvi in izdelanih krvnih pripravkov. Sodobne tehnologije omogočajo poleg omenjenegatudi avtomatizacijo laboratorijskega testiranja, zagotavljajo integriteto laboratorijskih izsledkov in omogočajo smotrnejšo izrabo krvi. Zaključki. Za večjo transfuzijsko varnost moramo s preventivnimi ukrepi in uvedbo hemovigilance nujno poskrbeti tudi v Sloveniji. Napake pri identifikaciji bolnika lahko preprečimo s sistemom zapestnic s črtno kodo za označitev bolnika in njegovih bioloških vzorcev administrativne napake v krvnibanki in laboratoriju pa z informacijskim sistemom in avtomatizacijo. Predvidevamo, da bo virtualni transfuzijski laboratorij postal sestavni del nove enotne slovenske transfuzijske mreže, ki bo pospešila, poenotila in poenostavila sedanje postopke v zvezi z naročanjem in aplikacijo krvnih pripravkov odgovornemu zdravniku bo omogočila izbrati optimalno shemo transfuzije, obenem pa bo omogočila spremljanje individualnih naročanj, odstopanj indikacij in diagnoz, kar je potrebno za analizo in izboljšavo kakovosti ter za analizo stroškov zdravjenja. Ti ukrepi predstavljajo tudi prvi nujni korak v posodobitvi transfuzijske medicine in s tem tudi slovenskega zdravstva v smislu zagotavljanja celostne kakovosti.

Starostne spremembe imunskega sistema so vzrok za zmanjšano sposobnost obrambe proti okužbam, za pojav vnetnih bolezni in povečano incidenco raka. Obstaja več teorij, ki skušajo razložiti številne istočasne in prepletene procese staranja. Po teoriji staranja matičnih celic je za staranje tkiv in organov odgovoren upad sposobnosti matičnih celic, da bi uspešno obnavljale tkiva. Namen naše raziskave je bil na modelu laboratorijskih miši raziskati, ali lahko presaditev celic kostnega mozga (KM) mladih živali izboljša delovanje imunskega sistema starih živali ter s tem izboljša njihovo splošno zdravje in morda celo podaljša življenjsko dobo. Neobsevanim samicam BALB/c smo v 14., 16. in 18. (19.) mesecu starosti presadili celice KM mladih samcev. Vsem prejemnicam celic KM smo določili stopnjo himerizma v KM, v izoliranih populacijah limfocitov T, limfocitov B in nevtrofilcev ter v CFU kolonijah, pripravljenih iz KM prejemnic. Analizirali smo spremembe števila celic prirojenega in pridobljenega imunskega sistema ter funkcij imunskih celic po presaditvi (sposobnost endocitoze, citokinski odziv in test stimulacije proliferacije). Manjše število živali smo imunizirali s hemocianinom iz školjke (KLH) ter z analizo titra protiteles testirali njihov imunski odziv na antigen. Pri vseh miših smo redno ocenjevali 30 različnih parametrov ter določili indeks krhkosti. Spremljali smo njihovo življenjsko dobo ter ob smrti opravili raztelesbe ter beležili patoanatomske spremembe. Rezultati so pokazali, da so imele stare miši, ki so prejele celice mladega KM, izboljšane nekatere imunske parametre prirojenega in pridobljenega imunskega sistema v primerjavi s kontrolno skupino, vendar teh ni bilo dovolj, da bi lahko hipotezo raziskave potrdili. Prejemnice KM niso imele vidno boljšega splošnega zdravja, indeks krhkosti ni pokazal razlik med poskusno in kontrolno skupino in miši tudi niso živele dlje od kontrolne skupine miši. Age-related changes in immune functions are the cause of the increased vulnerability to infections, inflammatory diseases, and cancer incidence. There are many existing theories that try to explain the numerous, simultaneous, and interconnected aging processes. The stem cell theory of aging postulates that stem cells become inefficient at maintaining the original functions of the tissues. In our study, we used a mice model to observe whether non-conditioned bone marrow (BM) cell transplantation from young donors to old recipients would improve their immune system functions, improve their general health, and possibly even extend their lifespans. Female BALB/c mice received BM cells from young donors at 14, 16 and 18(19) months of age. We determined chimerism levels in the BM, isolated T cell, B cell and neutrophil populations, and in colony-forming units (CFU) in BM. We analysed the changes of relative cell number of the innate and adaptive immune systems, and conducted functional assays (endocytic capability, cytokine response, and cell proliferation). We immunized a small group of mice with keyhole limpet haemocyanin (KLH) antigen and tested their immune response to antigen. All mice were regularly assessed using 30 parameters to calculate a frailty index (FI). We noted their lifespans, conducted post-mortem examinations, and recorded any pathoanatomical changes. Our results showed that old mice, transplanted with the BM cells of young donors, had some improvement in the immune parameters of their innate and adaptive systems, however, the evidence was insufficient for us to confirm our hypothesis. Recipient mice did not experience a noticeable improvement in their general health, and the FI showed no significant differences between the transplanted and control mice. In addition, the transplanted group did not live longer than the control group.

Staranje je zapleten, več faktorski proces, ki prizadane celice, tkiva in organe. S staranjem organizma se stara tudi imunski sistem, ki je sicer ključen za obrambo organizma pred škodljivim okoljem. S starostjo povezane okvare imunskega sistema vodijo v nastanek večine s starostjo povezanih bolezni kot so rak, avtoimunske bolezni in srčno-žilne bolezni ter povročijo povečano dovzetnost posameznika za okužbe. Ena izmed možnih terapij, ki bi lahko izboljšala delovanje imunskega sistema v starosti, je presaditev mladih krvotvornih matičnih in progenitorskih celic (KMPC), ki bi jih posamezniku odvzeli in shranili v njegovi mladosti ter ponovno presadili v njegovi starosti. Sklepamo, da bi na ta način mlade in zdrave KMPC po presaditvi »pomladile« stari imunski sistem ter izboljšale njegovo delovanje, ob enem pa bi posamezniku podaljšali njegovo življensko dobo. Omenjeni princip se imunuje heterohrona avtologna presaditev mladih krvotvornih matičnih celic. V raziskavi smo v mišjem modelu uporabili nov način presaditve KMPC s predhodno mobilizacijo endogenih KMC v periferno kri. V ta namen smo uporabili molekulo CASIN, ki začasno izprazni nišo kostnega mozga prejemnika, na ta način pa omogoči večjemu številu presajenih celic pritrjanje vanjo. Z uporabo molekule CASIN smo po presaditvi mladih KMPC starim prejemnicam dosegli visok odstotek donorskega himerizma, dokazali nekatere izboljšave v imunskem sistemu in rahlo povišali mediano preživetja. Kljub temu pa bi bilo za prenos v humano medicino potrebno izvesti dodatne raziskave, ki bi potrdili ugodne učinke tovrstne terapije. Aging is a complex multi-factorial process that affects cells, tissues and organs. It also affects the immune system, which is crucial for the body's defense against the harmful environment. Age-related defects of the immune system lead to the development of most age-related diseases, such as cancer, autoimmune and cardiovascular diseases and cause an increased susceptibility to infections. One of the strategies that could improve the function of the immune system of an old individual is the transplantation of young hematopoietic stem and progenitor cells (HSCs) which would be collected from an individual in his/her youth and cryopreserved for a long time. After re-infusion later in life of the same individual, young and healthy HSPCs could »rejuvenate« the old immune system and improve its functioning. Furthermore, individuals lifespan could be prolonged. The process is called heterochronous autologous hematopoietic stem cell transplantation. In this study we used a new method of HSC transplantation with prior mobilization of endogenous HSCs into peripheral blood. For this purpose, we used the molecule CASIN, which temporarily empties the recipient's niche, thus allowing engraftment of a larger number of transplanted cells. With the use of the CASIN molecule, we achieved a high percentage of donor chimerism after transplantation of young HSPCs into old recipients. In the recipients of young HSPCs, we detected some improvements in the function of the immune system and a slighty higher median lifespan. Nevertheless, for translation into human medicine, additional research would be needed to confirm the beneficial effects of this type of therapy.

Presaditev alogenskih krvotvornih matičnih celic je način zdravljenja določenih malignih in nemalignih bolezni. Stanje po alogenski presaditvi, pri katerem so v prejemnikovem telesu sočasno prisotne celice prejemnika in darovalca krvotvornih matičnih celic, imenujemo himerizem. Z določitvijo himerizma lahko zgodaj zaznamo zaplete kot so ponovitev bolezni, bolezen presadka proti gostitelju in zavrnitev presadka. Sledenje himerizma je zato pomembno za oceno in napoved uspešnosti presaditve krvotvornih matičnih celic in omogoča hitrejše ukrepanje ter poveča možnost ozdravitve bolnika. V nalogi smo za sledenje himerizma po presaditvi krvotvornih matičnih celic uporabili metodo PCR v realnem času in ocenili primernost kompleta setov AlleleSEQR (Celera, ZDA) ter programske opreme AlleleSEQR Suite v1.1 Chimerism Analysis Software (Celera, ZDA) in KMRengine Chimerism Analysis Software (GenDx, Nizozemska). Za testiranje smo uporabili vzorce genomske DNA, izolirane iz periferne krvi 23 bolnikov in njihovih 23 darovalcev. Pri vseh parih smo med 34 bialelnimi polimorfizmi insercij/delecij določili informativne označevalce, ki omogočajo razlikovanje med prejemnikovo in darovalčevo genomsko DNA. Nato smo s testom za kvantitativno določanje himerizma v več zaporednih vzorcih prejemnika, odvzetih v različnih časovnih intervalih po presaditvi krvotvornih matičnih celic, določili delež prejemnikove genomske DNA. Rezultate določitve himerizma smo primerjali s klinično sliko bolnika. Informativnost nabora označevalcev je bila 100 % za vse pare prejemnikov in darovalcev ter je bila po pričakovanju za nesorodne pare večja kot za sorodne. Pri 19 od 23 bolnikov se je klinična slika popolnoma ujemala z rezultati določitve himerizma. Pri štirih bolnikih se klinična slika ni vedno ujemala z rezultatom določitve himerizma. Pri petih bolnikih bi bilo za popolno interpretacijo rezultatov določitve himerizma potrebnih več zaporednih vzorcev. Bolezen presadka proti gostitelju je bila prisotna pri večjem deležu bolnikov s popolnim himerizmom kot pri bolnikih z mešanim himerizmom. Občutljivost metode je bila visoka, saj smo zaznali že 0,02 % himerizem, kar daje zgodnjo informacijo o dinamiki deleža prejemnikove genomske DNA. S testnim sistemom smo uspešno opravili tudi zunanjo kontrolo kakovosti. Zaključimo lahko, da je testni sistem primeren za sledenje himerizma po presaditvi alogenskih krvotvornih matičnih celic pri slovenski populaciji prejemnikov krvotvornih matičnih celic. Allogeneic hematopoietic stem cell transplantation is used in treating certain malignant and non-malignant diseases. Chimerism after allogeneic haematopoietic stem cell transplantation is a condition, in which recipient and donor cells are present in recipient’s body. Chimerism analysis enables early detection of complications, such as disease relapse, graft-versus-host disease and graft rejection. Chimerism monitoring is therefore important for assessment and prediction of successfulness of hematopoietic stem cell transplantation and enables earlier intervention and thus the possibility of a patient being cured is higher. In our study we determined the usefulness of the AlleleSEQR (Celera, USA) kit and software AlleleSEQR Suite v1.1 Chimerism Analysis Software (Celera, USA) and KMRengine Chimerism Analysis Software (GenDx, Netherlands) for chimerism monitoring after hematopoietic stem cell transplantation with the use of quantitative PCR. Genomic DNA samples isolated from peripheral blood of 23 patients and their 23 donors were used for testing. Among 34 biallelic insertion/deletion polymorphisms informative markers were determined, which enables differentiation between donor and recipient genomic DNA. The proportion of recipient genomic DNA in several successive recipient samples, collected in different time intervals after transplantation, was then determined using the test for quantitative chimerism monitoring. The chimerism results were compared with patient’s clinical picture. The informativity of the assay was 100 % for all included donor/recipient pairs and was higher for unrelated pairs than for related. The results of the determination of proportion of genomic DNA matched with the patient’s clinical picture in 19 patients out of 23. The chimerism results did not always match with the clinical picture in four patients. In five patients we would need more successive samples to completely interpret the chimerism results. A smaller proportion of patients with mixed chimerism had graft-versus-host disease compared to the patients with complete chimerism. The sensitivity of the assay is high, as we detected 0,02 % chimerism, which enables earlier information about the change in proportion of recipient genomic DNA. The external quality control was successfully passed with the tested system. We can conclude that the test system is suitable for chimerism monitoring after allogeneic haematopoietic stem cell transplantation for Slovenian population of haematopoietic stem cell recipients.

Mezenhimske stromalne celice (MSC) so eden glavnih virov za celične terapije in tkivno inženirstvo. Sposobne so samoobnavljanja in diferenciacije, lahko uravnavajo imunski odziv in sodelujejo pri celjenju poškodb. S staranjem se njihove regenerativne sposobnosti zmanjšajo. Pri terapijah z MSC se zato poraja vprašanje, kako starost darovalca in celic vpliva na izid zdravljenja. V magistrskem delu smo skušali ugotoviti, kako starost miši BALB/c vpliva na lastnosti njihovih MSC. S pretočno citometrijo smo spremljali izražanje celičnih označevalcev med gojenjem mladih in starih celic, spremljali smo proliferacijo celic s testom MTT, določali smo sposobnost migracije celic s testom celjenja ran in prisotnost senescentnih celic v kulturi. Ugotovili smo, da se mlade celice hitreje delijo, imajo nižji delež senescentnih celic v kulturi in slabše migracijske sposobnosti v primerjavi s starimi celicami. Med izražanjem celičnih označevalcev med skupinama nismo opazili razlik. Mesenchymal stromal cells are one of the main sources for cell therapy and tissue engineering. They are capable of self-renewal and differentiation. They can regulate a body's immune response and participate in its wound-healing process. Their regenerative abilities reduce as they age. MSC therapies therefore raise a question of how the age of the donor affects the outcome of the treatment. In this master's thesis, we tried to determine how the age of BALB/c mice affects the properties of their MSC. We monitored the expression of cell markers during cell culture with flow cytometry. We monitored cell proliferation rate with the MTT assay and analyzed mouse MSC's migration capacities with wound-healing assay. By measuring the β-galactosidase activity we determined the presence of senescent cells in culture. We discovered that young cells divide more rapidly, have a lower proportion of senescent cells in culture and have lower migration capacity when compared to older cells. The expression of cell markers is the same for both cell groups.