title: The applicability of different PCR-based methods for fetal RHD and K1 genotyping: a prospective study.
abstract: The applicability of different PCR-based assays for fetal RHD and K1 genotyping using DNA isolated from uncultured amniotic fluid cells has been tested prospectively: cord blood serotyping served as a control. For RHD genotyping, DNA was amplified with PCRs specific for RHD exon 7, the 3'-non-coding region and intron 4, using standard conditions. The results of these three separate assays were compared to those of a newly-developed multiplex PCR, simultaneously amplifying six regions of RHD. The PCRs analysing the 3'-non-coding region or intron 4 often yielded false-negative results or no results at all. Results of the exon 7 PCR and of the multiplex PCR always corresponded with postnatal serotyping, the multiplex PCR having the advantage of analysing six RHD-specific exons simultaneously. For K1 genotyping, two different PCR-based assays, both analysing the presence of T578C in the KEL gene, were applied. With the first method, a consensus 740-bp product of the KEL gene was amplified and subsequently specifically digested. As we were not able to obtain any PCR product from amniotic fluid DNA, we developed a new K1-specific PCR, amplifying a fragment of 91 bp only in cases of K1-positivity. With this PCR, all K1 genotyping results (n=30) correctly predicted the phenotypes. We conclude that fetal RHD and K1 genotyping can be performed reliably with DNA from uncultured amniotic fluid cells.
authors.affiliation: Central Laboratory of the Netherlands Red Cross Blood Transfusion Service and Laboratory of Experimental and Clinical Immunology, Academic Medical Center, University of Amsterdam, Plesmanlaan 125, 1066 CX Amsterdam, The Netherlands.
authors.forename: B H
authors.initials: BH
authors.lastname: Faas
authors.forename: P A
authors.initials: PA
authors.lastname: Maaskant-Van Wijk
authors.forename: A E
authors.initials: AE
authors.lastname: von dem Borne
authors.forename: C E
authors.initials: CE
authors.lastname: van der Schoot
authors.forename: G C
authors.initials: GC
authors.lastname: Christiaens
mesh_terms: D000653:Amniotic Fluid / Q000737:chemistry; D004247:DNA / Q000032:analysis* / Q000302:isolation & purification; D004899:Erythroblastosis, Fetal / Q000235:genetics / Q000276:immunology; D005260:Female; D005838:Genotype; D006801:Humans; D007231:Infant, Newborn; D007626:Kell Blood-Group System / Q000235:genetics*; D016133:Polymerase Chain Reaction; D011247:Pregnancy; D011296:Prenatal Diagnosis; D011446:Prospective Studies; D012204:Rh-Hr Blood-Group System / Q000235:genetics*
chemical_list: D007626:Kell Blood-Group System; D012204:Rh-Hr Blood-Group System; D004247:DNA