98 KB21 KB192920262004Blejec, TanjaBricl, IrenaDovč, TadejaRožman, Primožstr. I-139-I-142ISSN:1318-0347COBISSID_HOST:17683417URN:URN:NBN:SI:doc-9YDHP0MDslSlovensko zdravniško društvoZdravniški vestnikBlood Grouping And CrossmatchingDnaEritroblastoza plodaErythroblastosis, FetalGenotipGenotypeKrvne skupine in navzkrižno določanjeNosečnostPolimerazna, verižna reakcijaPolymerase Chain ReactionPredrojstvena diagnostikaPregnancyPrenatal DiagnosisRh-Hr Blood-Group SystemRh-Hr sistem krvnih skupinDoločanje plodove krvne skupine med nosečnostjo| Determination of fetal blood group in pregnancy|Background. Prenatal diagnostics with the genotyping using the PCR analysis ofthe fetal DNA holds an important place in modern gynaecology. Lately, noninvasive fetal genotyping became feasible after fetal DNA was proven and isolated from the peripheral blood of the mother. Methods. Peripherral blood samples of ninety-six RhD negative women in their 23-33 week of pregnancy werecollected and the DNA of the fetus isolated with the modified method of Finning et al Fetal RHD genotype was than determined with real time PCR methodusing the ABI Prism 7900 Sequence Detection System. The presence of the three parts of the RHD gene, intron 4, exon 7 as well as 10, was assesed. In order to confirm the presence of fetal DNA, the presence of the SRY gene was used with male fetuses. If the PCR reaction results of the RHD and SRYgene were negative, we assumed that the fetus was a female whose RhD was negative. In order to verify that, a presence of eight chosen polymorphic alleles was determined from the the DNA samples of the mother and from her plasma, thus indirectly confirming that it was the DNA of the fetus which was being tested.Results. In this research we accuratly predicted the genotype and gender of the fetuses of 96 RhD negative pregnant women from their peripheral blood. The predicted results of the research were compared with the actual gender and RhD phenotype of the newborn, that were determined at birth. Our accuracy in predicting the gender and the presence of the RHD gene using our real-time PCR method was 100%. Conclusions. We anticipate the fetal genotype, determined on the basis of the fetal DNA being present in the peripheral bloodof the mother, to take on a leading role in prenatal investigations, especially in cases of RhD immunisation during pregnancy and in prevention of RhD immunisation with anti-D immunoglobulin in the 28th week of pregnancyIzhodišča. Prenatalna diagnostika z genotipizacijo plodove deoksiribonukleinske kisline (DNK) zavzema pomembno mesto v modernem porodništvu. V zadnjem času je možno iz venske krvi matere osamiti plodovo DNK, kar omogoča neinvazivno prenatalno genotipizacijo. Material in metode. V 23. do 33. tednu nosečnosti smo 96 RhD-negativnim nosečnicam odvzeli vensko kri, iz nje osamili plodovo DNK s prilagojenim načinom po Finningu s sod. in določili plodov genotip RHD. Detekcijo gena RHD smo izvedli z metodo PCR v realnem času na instrumentu ABI Prism 7900 Sequence Detection System. Določalismo prisotnost treh delov genskega zapisa gena RHD, tj. introna 4, eksona 7 in eksona 10. Za potrditev, da gre za plodovo DNK, smo pri moških plodih sklepali na podlagi prisotnosti gena SRY. Če sta bila rezultata PCR reakcije za gena RHD in SRY negativna, smo sklepali, da nosi nosečnica plod ženskega spola, ki je RhD-negativen. V teh primerih smo za potrditev, da gre za plodovo DNK, določali še prisotnost osmih naključnih polimorfnih alelov iz vzorca DNK matere in vzorca DNK pIoda iz materine plazme in tako posredno potrdili, da je šlo za plodovo DNK. Rezultati. V raziskavi smo 96-im RhD-negativnim nosečnicam iz vzorca venske krvi pravilno napovedali plodov genotip RHD in njegov spol. Napovedane rezultate preiskav smo primerjali z dejanskim spolom in fenotipom RhD novorojenčka, ki smo ju določili po rojstvu.Natančnost napovedi spola in prisotnosti gena RHD z našo metodo PCR v realnem času je bila 100%. Zaključki. Pričakujemo, da bo določitev plodovega genotipa na podlagi proste DNK v periferni krvi nosečnice zavzela pomembno mesto v prenatalnih preiskavah, še posebno v primerih imunizacije na RhD antigen med nosečnostjo in pri izvajanju predporodne preventive z imunoglobulinom antiD v 28. tednu nosečnostiTEXTznanstveno časopisjejournalsSlovenian National E-content AggregatorNational and University Library of SloveniaSlovensko zdravniško društvo