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La celiaquía es una enfermedad autoinmune que afecta al intestino delgado como resultado de una respuesta inmunitaria frente a la ingesta de gluten, el cual está presente en cereales como el trigo, la cebada, la avena o el centeno. En general, esta ingesta da lugar a la aparición de síntomas digestivos que solo se pueden mitigar siguiendo una dieta sin gluten. La enfermedad celíaca se manifiesta en individuos genéticamente predispuestos que presentan antígenos leucocitarios humanos de clase II (HLA-II), principalmente HLA-DQ2 (90%), y con menor frecuencia HLA-DQ8 (5%-10%). En la última década, la celiaquía se ha considerado una enfermedad rara, pero hoy en día se sabe que está distribuida universalmente, siendo una de las enfermedades genéticas más conocidas. Tiene una prevalencia media del 1%-2% en la población general, pero está claramente subestimada e infradiagnosticada a nivel mundial. Además, su frecuencia está aumentando en muchos países en desarrollo debido a la occidentalización de la dieta, a los cambios en la producción y a la preparación del trigo, y una mayor concienciación sobre la enfermedad. En el Hospital General Universitario Dr. Balmis de Alicante se realiza el estudio del HLA asociado a celiaquía mediante la PCR basada en sondas oligonucleotídicas específicas de secuencia (PCR-SSO). Sin embargo, recientemente se ha comercializado un nuevo kit basado en PCR en tiempo real (qPCR) que permite detectar los alelos asociados a celiaquía en menor tiempo y a menor coste. El objetivo del presente trabajo es realizar una revisión bibliográfica actualizada de la enfermedad celíaca y validar el nuevo kit de qPCR (Coeliac ImmuneKit) para sustituir a la PCR-SSO en el estudio del HLA asociado a enfermedad celíaca. Para esto último, mediante el programa estadístico SPSS se han obtenido tablas de contingencia para conocer la concordancia con el índice kappa o de Cohen, y los valores de sensibilidad y de especificidad de este nuevo kit de qPCR en comparación con el genotipo de referencia resultante de las técnicas basadas en PCR-SSO, en este caso el Inmuno LiPA y el Mr. Spot. Los resultados obtenidos se comparan con otros análisis diagnósticos, como son los serológicos u histológicos, y si éstos son precisos y fiables se concluye que este nuevo kit comercial de qPCR (Coeliac ImmuneKit) es válido para implementarlo en la Sección de Inmunología del Servicio de Análisis Clínicos del Hospital General Universitario Dr. Balmis de Alicante, sustituyendo así las anteriores técnicas de tipaje basadas en PCR-SSO.

Sustav HLA najsloženiji je genetski sustav kod čovjeka. Između ostalog, osobitosti ovog sustava su izraziti polimorfizam, kao i povezanost ovog sustava s bolestima, što je od izrazite važnosti u dijagnostici autoimunih bolesti kao i neuroloških oboljenja. Migrena je ponavljajuća, umjereno jaka do vrlo jaka glavobolja u trajanju između 4 i 72 sata u slučaju kada se ne liječi. Postoji nekoliko čimbenika rizika povezanih s razvojem migrene. U nepromjenjive čimbenike spadaju genetika, dob i spol. Cilj je ovog rada odrediti zastupljenost alela sustava HLA u pacijenata kojima je dijagnosticirana migrena i kod pacijenata koji pate od jakih glavobolja na području Splitskodalmatinske županije kao i prikazati zastupljenost pojedinih alela lokusa HLA - DRB1*. Kod ispitanika s migrenom i jakim glavoboljama koji su imali HLA-DRB1*16, zastupljenost spolova pokazuje da je znatno veći postotak oboljelih kod žena (68.96%) u odnosu na muškarce (31.04%). Najveći broj ispitanika je bio u dobnoj skupini od 46 do 65 godina. Antigeni i aleli sustava HLA za potrebe ove studije određeni su serološkim i molekularnim metodama. Antigeni sustava HLA određuju se testom mikrolimfocitotoksičnosti (MLCT), dok su za molekularno određivanje alela lokusa sustava HLA korištene metode PCR-SSO i PCR-SSP. Određivanjem alela lokusa HLA-DRB1 u skupini ispitanika s migrenom, uočeno je 14 različitih alela, od kojih najveću zastupljenost imaju HLA-DRB1*16 (19,59%) te HLADRB1*11,-13,-15 s učestalosti od 10,14%. U usporedbi sa kontrolnom skupinom, utvrđeno je da je učestalost alela HLA-DRB1*16 (19,59% vs 9,60%, p=0,0063) i HLA-DRB1*10 (2,7% vs 0%, p=0,0327) statističko značajno povećana u skupini pacijenata koji boluju od migrene ili jake glavobolje. Budući da su našim ispitanicima u dijagnostičke svrhe određivani i aleli lokusa HLA razreda I (HLA-A, HLA-B) prilikom analize uočena je statistički značajna povezanost alela HLA-B*57 kod pacijenata s migrenom i jakim glavoboljama u odnosu na kontrolnu skupinu zdravih ispitanika. Da bismo sa sigurnošću govorili o povezanosti migrene i jakih glavobolja i alela HLADRB1*16 i ostalih alela lokusa sustava HLA (HLA-A, HLA-B) svakako je potrebno proširiti studiju na veći broj ispitanika. The HLA system is the most complex genetic system in humans. Among other things, the peculiarities of this system are its pronounced polymorphism, as well as the connection of this system with diseases, which is of great importance in the diagnosis of autoimmune diseases as well as neurological diseases. Migraine is a recurrent, moderately severe to very severe headache lasting between 4 and 72 hours if left untreated. There are several risk factors associated with developing migraines. Immutable factors include genetics, age and gender. The objective of this work was to determine the representation of alleles of the HLA system in patients suffering from migraines and severe headaches in the Split-Dalmatia County, as well as to show the representation of individual alleles of the HLA locus - DRB1*. In subjects with migraine and severe headaches who had HLA-DRB1*16, the representation of genders shows that the percentage of patients is significantly higher in women (68.96%) compared to men (31.04%). The largest number of respondents was in the age group of 46 to 65 years. Antigens and alleles of the HLA system for the purposes of this study were determined by serological and molecular methods. Antigens of the HLA system are determined by the microlymphocytotoxicity test (MLCT), while the PCR-SSO and PCR-SSP were used for the molecular determination of alleles of the HLA system locus. . By determining the alleles of the HLA-DRB1 locus in the group of subjects with migraine, 14 different alleles were observed, of which the highest prevalence is HLA-DRB1*16 (19.59%) and HLA-DRB1*11,-13,-15 with a frequency of 10 , 14%. Compared to the control group, the frequency of alleles HLA-DRB1*16 (19.59% vs 9.60%, p=0.0063) and HLA-DRB1*10 (2.7% vs 0%, p =0.0327) statistically significantly increased in the group of patients with migraine or severe headache. Since alleles of the HLA class I locus (HLA-A, HLA-B) were also determined for diagnostic purposes in our subjects, the analysis revealed a statistically significant association of the HLAB*57 allele in patients with migraine and severe headaches compared to the healthy control group examinee. In order to talk with certainty about the connection between migraine and severe headaches and the HLA-DRB1*16 allele and other alleles of the HLA system locus (HLA-A, HLA-B), it is certainly necessary to expand the study to a larger number of subjects.

Sustav HLA (humani leukocitni antigen) glavni je sustav tkivne podudarnosti. Ključnu ulogu u imunološkim procesima imaju molekule HLA odnosno membranski proteini koji djeluju kao antigeni i u stranom organizmu uzrokuju imunološku reakciju. Antigeni i geni sustava HLA određuju se serološkim i molekularnim metodama. Test mikrolimfocitotoksičnosti serološka je metoda koja se najčešće koristi za određivanje antigena sustava HLA razreda I i II. Molekularno određivanje HLA gena može se izvesti pomoću dvije metode, a to su metoda PCR-SSO (engl. Polymerase chain reaction Sequence Specific Oligonucleotide), a druga metoda je PCR-SSP (eng. Polymerase chain reaction – Sequence Specific Primer). Prije izvođenja molekularnih metoda potrebno je izvršiti izolaciju DNA iz periferne krvi. PCR-SSO metoda zasniva se se na povezivanju molekule DNA koja se amplificirala na suspenziji mikrosfera. Reumatoidni artritis kronična je, teška, autoimuna upalna bolest čija je glavna karakteristika sinovitis. Dolazi do razaranja vezivnoga tkiva i zglobnih struktura što u konačnici dovodi do bolnih i upalnih zglobova s čestim deformacijama. Cilj rada bio je odrediti učestalost alela sustava HLA kod ispitanika koji boluju od reumatoidnog artritisa na području Splitsko-dalmatinske županije. Molekularnim metodoma dokazuje se povezanost zajedničkog epitopa. Molekula HLA razreda II bila je područje od interesa jer je pokazivala posebnu osjetljivost na razvoj bolesti. Molekularne metode olakšale su proučavane te povezanosti koristeći nasljeđene sekvence od navedenih aminokiselina QKRAA, QRRAA ili RRRAA koje se nalaze u hipervarijabilnim regijama kao ciljna mjesta. The HLA system (human leukocyte antigen) is the major tissue matching system. A key role in immunological processes has HLA molecules and membrane proteins that act as antigens and the foreign organism causing an immune response. Antigens and genes of the HLA system are determined by serological and molecular methods. The microlymphocytotoxicity test is a serological method most commonly used to determine class I and II HLA system antigens. Molecular determination of the HLA gene can be performed using two methods, namely the PCR-SSO method (Polymerase chain reaction Sequence Specific Oligonucleotide), and the other method is PCR-SSP (Polymerase chain reaction - Sequence Specific Primer). Before performing molecular methods, it is necessary to isolate DNA from peripheral blood. The PCR-SSO method is based on the binding of a DNA molecule that has been amplified on a suspension of microspheres. Rheumatoid arthritis is a chronic, severe, autoimmune inflammatory disease whose main feature is synovitis. There is destruction of connective tissue and joint structures which ultimately leads to painful and inflammatory joints with frequent deformities. The aim of this study was to determine the frequency of HLA alleles in subjects suffering from rheumatoid arthritis in the Split-Dalmatia County. Molecular methods prove the connection of a common epitope. The HLA class II molecule was an area of interest because it showed particular susceptibility to disease development. Molecular methods have facilitated the study of these linkages using inherited sequences from the listed amino acids QKRAA, QRRAA or RRRAA found in hypervariable regions as target sites.

Background: Hepatic hydatid disease (HHD) is a parasitic zoonosis caused by larval stage of Echinococcus tape worm. In addition to environmental factors, genetic constitution of hosts seems to play a crucial role in acquiring the infection and developing disease. Aims: This study was carried out to investigate the association of HLA-class I and class II (A, B, DR and DQ) alleles with HHD by genotyping in Iraqi patients, as well as to provide information about genotypes that confer susceptibility or resistance to develop the disease. Materials and Methods: Twenty patients with HHD, their age range (20-50) years and 20 healthy controls their ages were matched with the patients were enrolled in this study. Blood was collected from patients and controls, DNA was extracted from blood samples, and then HLA-class I and class II genotyping was performed by polymerase chain reaction-sequence specific oligonucleotide probes (PCR-SSO). Results: The present findings showed that frequencies of HLA-A*32 (65%; P=0.011), DRB1*11 (60%; P=0.004) and DQB1*03 (70%; P=0.007) alleles are significantly higher in patients than controls, while the frequency of DRB1*04 was significantly decreased in patients when compared to controls (25% vs. 75%; P=0.002). Furthermore, the current study could not observe significant differences in the frequencies of HLA-B alleles between patients and controls. Conclusions: We concluded that HLA-A*32, DRB1*11 and DQB1*03 alleles might contribute to the increased susceptibility to HHD and DRB1*04 could be a protective marker against the disease.

Cilj rada Cilj rada je objasniti povezanost sustava HLA i celijakije te istražiti kolika je učestalost alela lokusa HLA-DQA i DQB kod pacijenata oboljelih od celijakije. U provedenom istraživanju cilj je bio opisati vezu između alela HLA-DQB1*03:01 i HLA-DQB1*03:02. Materijali i metode Ovo istraživanje provedeno je u Laboratoriju za tipizaciju (Zavod za transfuzijsku medicinu, KBC Split) te je obuhvatilo 249 pacijenata podijeljenih u dvije skupine. Ciljnu skupinu s 199 pacijenata s sumnjom na celijakiju te kontrolnu skupinu od 50 ispitanika zdrave populacije Splitsko- dalmatinske županije. U svrhu genetskog testiranja korištene su metode izolacije DNA vršene pomoću komercijalnog kita firme Roche Diagnostics (High Pure PCR Template Preparation Kit), te metoda PCR-SSO (engl. Polymerase chain reaction-Sequence Specific Oligonucleotids). Učestalost alela lokusa HLA-DQA1 i HLA-DQB1 sustava HLA određena je direktnim brojanjem. Za usporedbu razlike između ispitivane i kontrolne skupine korištena je 2x2 tablica a za vrijednosti manje od 5 korišten je Fisher-ov egzaktni test (statistički značajnim rezultatom smatran je svaki kojem je vrijednost P

Uvod: Glavni sustav tkivne snošljivosti kod čovjeka, nazvan sustav HLA, jedan je od najistraživanijih genskih sustava. Smješten je na kraćem kraku 6. kromosoma i čine ga antigeni i aleli razreda I, II i III. Zbog izrazitog polimorfizma od iznimnog je značaja u transplantacijskoj medicini, populacijskoj genetici i dijagnostici raznih bolesti. Glavna je uloga sustava HLA razlikovanje vlastitih stanica od tuđih te pokretanje adekvatnog imunološkog odgovora pri kontaktu organizma sa stranim antigenima. Autoimune bolesti nastaju kada dođe do zatajenja te osnovne funkcije imunološkog sustava, odnosno kada organizam na vlastite antigene počne reagirati kao na tuđe. Brojni geni sustava HLA u uskoj su korelaciji s pojavom autoimunih bolesti, poput celijakije, dijabetesa tipa I, bolesti štitnjače i više reumatskih bolesti. Cilj rada: Prikazati iznimnu važnost sustava HLA u dijagnostici te njegovu povezanost s raznim bolestima među kojima su i autoimune bolesti (reumatoidni artritis, psorijatični artritis, sistemska skleroza, celijakija i brojne druge). Metode: Laboratoriji za tipizaciju tkiva za određivanje gena i antigena sustava HLA koriste serološke metode i metode molekularne biologije. Za određivanje antigena sustava HLA serološkom metodom koristi se test mikrolimfocitotoksičnosti (MLCT), dok se za određivanje alela sustava HLA molekularnim metodama koriste metode PCR – SSP (lančana reakcija polimerazom s početnicama specifičnih sekvenci) ili PCR – SSO (lančana reakcija polimerazom s oligonukleotidima specifičnih sekvenci. Zaključak: Određivanje antigena i alela sustava HLA u pacijenata s reumatološkim oboljenjima od iznimne je važnosti u dijagnostici i terapiji pacijenta. Usavršavanjem metoda molekularne biologije svakodnevno se dolazi do novih spoznaja o povezanosti sustava HLA i bolesti. Introduction: The major histocompatibility complex, named the human leukocyte antigen system, is one of the most researched genetic systems. It is located on the short arm of the 6th. chromosome and it is made of antigens and alleles of class I, II and III. Being highly polymorphic, it is of exceptional importance in fields of transplantation medicine, population genetics and in diagnostics of various diseases. The main role of the HLA system is differentiation between organism's own cells and foreign cells and an adequate initiation of the immune response when in contact with foreign antigens. Autoimmune diseases develop when it comes to failure of the main role of immune system, that is, when an organism fails to recognise its own antigens and starts to react them as to foreign ones, attacking them. Numerous genes of the HLA system are tightly correlated with the development of autoimmune diseases like celiac disease, type I diabetes, thyroid diseases and a number of rheumatic diseases. Objective: To interpret the extreme importance of the HLA system in disease diagnostics and its correlation to various diseases including autoimmune diseases (rheumatoid arthritis, psoriatic arthritis, systemic sclerosis, celiac disease). Methods: To determine genes or antigens of the HLA system, tissue typing laboratories use both serological methods and methods of molecular biology. For determination of HLA antigens by serological method the microlymphocitotoxicity test is used (MLCT), while molecular methods for determining the alleles of HLA systems are PCR – SSP (Polymerase Chain Reaction with Specific Sequence Primers) and PCR – SSO (Polymerase Chain Reaction with Specific Sequence Oligonucleotide). Conclusion: The determination of antigens and alleles of the HLA system in patients with rheumatoid diseases is of exceptional value both in diagnostics and patient's treatment. With the development of molecular biology methods, new findings about the HLA system and its correlation to diseases arise daily.

Intermediate-resolution HLA-DQ typing has gained importance in organ transplantation recently. We evaluated the performance of the LIFECODES HLA-DQB1 typing kit (Immucor, USA) using sequence-specific oligonucleotide (SSO) probe and Luminex platform (Luminex Corp., USA) on 100 samples tested by sequence-based typing (SBT) using the AlleleSEQR HLA-DQB1 kit (Abbott Molecular, USA) in Korean individuals. No sample showed ambiguity in the assignment of 4-digit HLA-DQB1 allele with the LIFECODES HLA-DQB1 SSO typing kit, and the results were fully concordant with those of high-resolution typing of AlleleSEQR HLA-DQB1 SBT up to 4-digit level. Three samples required adjustment of false reactions (3/100, 3.0%): two samples with DQB1(*)03:03/(*)06:01 showed false-positive result in probe 253, and 1 sample with DQB1(*)04:02/(*)05:02 showed false-negative result in probe 217. We tested an additional sample with DQB1(*)03:03/(*)06:01, which showed same false-positivity in probe 253 and 2 samples with DQB1(*)04:02/(*)05:02, which showed no false reaction. The false reactions did not result in ambiguity or change in the HLA allele assignment. We could assign HLA-DQB1 alleles to 4 digit-level without ambiguity, with 100% concordance with the SBT results. Thus, LIFECODES HLA-DQB1 SSO typing kit showed good performance for intermediate-resolution HLA-DQB1 typing in clinical laboratory for organ transplantation in Koreans.

Uvod: Krvotvorne matične stanice (KMS) su ishodište svim krvnim stanicama organizma. Osobi oboljeloj od teških hematoloških bolesti transplantacijom KMS potrebno je nadoknaditi vlastite nefunkcionalne matične stanice. Tri glavna izvora matičnih stanica su: koštana srž, periferna krv te krv iz pupkovine odnosno umbilikalna krv. Transplantacije se dijele na: alogenu, autolognu te transplantaciju KMS iz umbilikalne krvi. Prije same transplantacije potrebno je odrediti primateljeve i davateljeve gene i antigene sustava HLA kao najsloženijeg genetskog sustava kod čovjeka Za uspješnu alo-transplantaciju potrebno je da podudarnost alela lokusa HLA između primatelja i davatelja bude 10/10 (HLA-A, -B, -C, -DRB1, -DQB1). Cilj: Cilj ovog rada je bilo objasniti ulogu sustava HLA u transplantaciji KMS te opisati i objasniti razlike između metoda koje se u tom procesu izvode. Materijali i metode: Metode koje se koriste za određivanje antigena i alela sustava HLA su: serološka metoda (test mikrolimfocitotoksičnosti) te metode molekularne biologije: PCR-SSP i PCR-SSO. Rezultati i rasprava: Otkrivanjem sustava HLA transplantacija KMS izbor je u liječenju mnogih hematoloških bolesti. Razvojem metoda određivanja gena i antigena sustava HLA povećana je uspješnost transplantacija krvotvornih matičnih stanica. Usavršavanje metoda molekularne biologije donosi i nove spoznaje o još većoj složenosti sustava HLA. Ključne riječi: transplantacija krvotvornih matičnih stanica, HLA, srodni i nesrodni davatelji, test mikrolimfocitotoksičnosti, PCR-SSP, PCR-SSO Introduction: Hematopoietic stem cells (HSCs) are the stem cells that give rise to all the other blood cells through the proces of haematopoiesis. Hematopoietic stem cell transplantation (HSCT) involves the intravenous infusion of autologous or allogeneic stem cells collected from bone marrow, peripheral blood, or umbilical cord blood to re-establish hematopoietic function in patients whose bone marrow or immune system is damaged or defective. This procedure is often performed as part of therapy to eliminate a bone marrow infiltrative process, such as leukemia, or to correct congenital immunodeficiency disorders. Before transplantation it is necessary to determinate HLA genes and antigens of recipients and donors. For successful allo- transplantation it is necessary to have 10 of 10 (HLA-A, -B, -C, -DRB1, -DQB1) allele HLA loci compatibility between recipient and donor. Goal: The aim of this thesis was to explain the role of HLA in transplantation of HSCs but also to describe and explain difference between the methods in use. Materials and methods: The methods used for the determination of antigens and alleles of HLA are: serological method (microlymphocitotoxicity test) and molecular biology methods: PCR-SSP and PCR-SSO. Results and discussion: After the discovery of HLA haematopoietic stem cells transplantation has become choice in treatment for many hemological diseases. The development of methods for gene and antigen HLA determination leads to increased efficacy in transplantation of HSC. Training methods of molecular biology bring new insights into the even greater complexities of the HLA. Key words: haematopoietic stem cell transplantation, HLA, related and unrelated donors, microlymphocitotoxicity test, PCR-SSP, PCR-SSO.

Receptores killer cell immunoglobulin-like (KIRs) são moléculas localizadas na superfície de células natural killer (NK) e em subpopulações de linfócitos T codificadas por genes do cromossomo 19q13.4. A interação entre receptores KIR e moléculas antígeno leucocitário humano (HLA) de classe I determina se células NK exercerão ou não sua função citotóxica e/ou secretora de citocinas ou se esta será inibida. Este trabalho teve por finalidade otimizar a metodologia para a genotipagem KIR, baseando-se nas condições descritas por Martin (2004). A técnica utilizada foi a reação em cadeia da polimerase com primers de sequência específica (PCR-SSP) com iniciadores sintetizados pela Invitrogen® e visualização do produto amplificado em gel de agarose a 2% com brometo de etídio. Adaptações foram realizadas e a concentração de alguns reagentes foi alterada, como a do controle interno de 100 nM para 150 nM, iniciadores específicos senso e antissenso de KIR12.5/12.3, KIR13.5/13.3, KIR14.5/14.3, KIR22.5/22.3 e KIR36.5/36.3 de 500 nM para 750 nM e da solução de MgCl2 de 1,5 mM para 2 mM. As concentrações dos demais reagentes e temperaturas de amplificação foram mantidas. Nessas condições, o uso da Taq DNA polimerase recombinante (Invitrogen®) foi satisfatório. Os resultados das genotipagens de 70 indivíduos foram confirmados por rSSO-Luminex® (One Lambda, Canoga Park, CA, EUA). A tipagem de genes KIR por essa técnica apresentou sensibilidade, especificidade, reprodutibilidade e baixo custo. The killer cell immunoglobulin-like receptors (KIRs) are molecules expressed on natural killer (NK) cells surface and in T-cell subsets encoded by genes located in chromosome 19q13.4. The interaction between KIR receptors and HLA class I molecules determines if the NK cells will fulfill their cytotoxic function and/or cytokine secretion or if this function will be inhibited. The objective of this work was to optimize KIR genotyping method described by Martin (2004). It was used PCR-SSP (polymerase chain reaction-sequence-specific primers) with primers synthesized by Invitrogen® and visualization of the amplified products on 2% agarose gel electrophoresis, containing ethidium bromide. Some adaptations were made and the reagents had their concentrations increased: the internal control from 100 nM to 150 nM, forward and reverse specific primers KIR12.5/12.3, KIR13.5/13.3, KIR14.5/14.3, KIR22.5/22.3 and KIR36.5/36.3 from 500 nM to 750 nM, and MgCl2 solution from 1.5 mM to 2 mM. Other reagent concentrations and amplification temperatures were maintained. Satisfactory results were obtained with Taq DNA Polymerase Recombinant (Invitrogen®). The results of seventy samples were confirmed by rSSO-Luminex® (One Lambda, Canoga Park, CA, USA). This KIR typing method proved to be accurate, specific, reproducible and cost effective.

Article, 信州医学雑誌 40(6): 553-565(1992)

Presajanje krvotvornih matičnih celic (KMC) se je v zadnjih desetletjih uveljavilo kot najuspešnejši način zdravljenja poprej večinoma neozdravljivih bolezni krvotvornih organov predvsem različnih vrst levkemij. Kadar bolniku ne najdemo glede na HLA enakega sorodnega darovalca, iščemo nesorodnega v registrih darovalcev krvotvornih matičnih celic. V strokovni literaturi najdemo dokaze, da neujemanja v tkivnih antigenih HLA-A, HLA-B, HLA-C in HLA-DR med darovalcem in prejemnikom KMC odločilno vplivajo na uspešnost presaditve oz. na preživetje bolnika po njej. Lokus HLA-C so med produkti genov HLA I. razreda odkrili najpozneje, tako da je njihov vpliv na izhod presaditve KMC manj raziskan. Ugotovili smo, da so za tipizacijo HLA-C najprimernejše metode, ki temeljijo na analizi DNK. Ker z eno metodo ne moremo razrešiti vseh dvoumnosti tipizacij, je trenutno po našem mnenju najboljša izbira kombinacija metod PCR-SBT in PCR- SSP. Antigene oz. alele HLA-C smo na vzorcu oseb slovenske narodnosti v različnih časovnih obdobjih tipizirali z metodama: PCR-SSO in PCR-SBT. PCR-SSP smo v obeh primerih uporabili le kot dodatno metodo. Za razliko od izbranih evropskih populacij, kjer je najpogostejši alel Cw*0701, je presenetljivo najpogostejši alel v slovenski populaciji Cw*0401. V primeru rekombinacij v antigenih HLA bolnik v družini nima primernega darovalca, prav tako je težka izbira oz. iskanje nesorodnega darovalca. Z namenom, da bi ugotovili pogostnost rekombinacij pri naših bolnikih smo analizirali 557 družin. Po določitvi haplotipov smo pri 2,7% družin opazili rekombinacijo. Ugotovili smo, da se pojavi v 40% primerov med lokusoma HLA-A in C, med lokusoma HLA-B in DRB1 pa v 60% primerov. Iz tega sklepamo, da neujemanja v HLA-C največ prispevajo k tkivni neskladnosti na lokusih HLA I. razreda. Razvidno je celo da ne gre za neujemanje v alelih HLA-C, pač pa za neujemanja v antigenih, kar predstavlja večjo oviro za presaditev KMC. Razlog je verjetno iskati v močnem vezavnem neravnovesju med lokusoma HLA-B in HLA-C, kar je razvidno tudi iz naše analize rekombinacij, ki jih med omenjenima lokusoma nismo opazili. Izsledki nekaterih študij kažejo na to, da obstaja veliko število različnih kombinacij antigenov HLA-B in HLA-C v vezavnem neravnovesju. Tako velikokrat pri darovalcu naletimo na drugačno kombinacijo antigenov HLA-B in HLA-C kot jo ima bolnik. Menimo, da bi hkratni izbor več darovalcev ter vključitev lokusa HLA-C k osnovni tipizaciji tako darovalca kot bolnika pripomogla k učinkovitejšemu in hitrejšemu zaključku preizkušanja tkivne skladnosti. During the past decades transplantation of haemapoietic stem cells has proven to be the most successful treatment method of diseases of haematopoietic organs such as different types of leukaemia, previously considered as mostly incurable. In case of unavailability of genotipicaly identical related donor, a search for an unrelated donor is initiated through bone marrow donor registries. Literature offers evidence on disparities at HLA-A, HLA-B, HLA-C and HLA-DR antigens between donor and recipient of haemapoietic stem cells, playing the most important role in outcome of allogenic stem cell transplantation and survival afterwards. HLA-C locus has been the last of HLA class I loci to be discovered, therefore the impact of its products on transplantation outcome has not been studied as extensively as others. DNA based analysis have shown to be the most adequate techniques. Using one technique only, many times gives ambiguous result. The best results are, in our opinion, achieved using the combination of both PCR-SBT and PCR-SSP typing technique. In our study blood samples, obtained from individuals of Slovene nationality, were typed at two different times using PCR-SSO and PCR-SBT technique. In both cases PCR-SSP was performed as an additional technique. The study yielded surprising results. The most common allele in Slovenian population is Cw*0401 as opposed to selected European populations with the highest frequency of Cw*0701 allele. Recombination between HLA genes in patient significantly reduces a possibility of finding a matched unrelated donor, whereas the possibility of finding the donor among relatives is excluded. In order to determine the frequency of recombinations among Slovenian patients 557 families were analysed. Determination of haplotypes showed 2,7% frequency of recombinations among families. While in 40% of the cases, recombination occured between HLA-A and HLA-C loci, in 60% of the case the recombination occured between HLA-B and HLA-DR loci. The study showed that out of HLA I loci disparities at HLA-C locus contribute the most to incompatibility. Disparities at HLA-C have not been observed at an allele level, but always at an antigen level representing significant obstacle regarding stem cell transplantation, which probably stems from linkage disequilibrium between HLA-B and HLA-C loci, that our study supports as no recombination between these two loci was detected. Due to many different possible combinations of HLA-B and HLA-C antigens, the same combination is rarely found in both the patient and the donor. It is of our opinion that the simultaneous typing of several potential donors and a mandatory inclusion of HLA-C locus in basic typing of both the patient and the donor contribute to more efficient and faster completion of the search

TEZ7190 Tez (Yüksek Lisans) -- Çukurova Üniversitesi, Adana, 2009. Kaynakça (s.59-61) var. ix, 62 s. : rnk.res. ; 29 cm. In this study, frequencies of HLA in our region have been determined. With this aim, the samples which were sent to Immunology Laboratory of the Center Laboratory Medicine Faculty of Çukurova University, were taken to be determined the types of HLA antigens. In this study, the datas of 771 person (339 patient, 432 donor) were analysed and the frequency of each lökosit antigen were calculated... Bu çalışmada, bölgemizde ki Human Lökosit Antijenlerinin (HLA) sıklığı araştırılmıştır. Bu amaçla; Çukurova Üniversitesi Tıp Fakültesi Balcalı Hastanesi Merkez Laboratuvarı immünoloji birimine HLA antijenlerini belirlemek amacıyla gönderilen örnekler değerlendirmeye alınmıştır. Çalışmamızda 339 hasta, 432 donör olmak üzere; toplam 771 kişinin verileri incelenmiş ve her bir lökosit antijenin sıklığı hesaplanmıştır. HLA antijenlerinin belirlenmesinde PCR-SSO (squence spesific oligonucleotid) yöntemi kullanılmıştır. Bu çalışma Ç.Ü. Bilimsel Araştırma Projeleri Birimi Tarafından Desteklenmiştir. Proje No:TF2008YL3