title: Prevalence of RhD status and clinical application of non-invasive prenatal determination of fetal RHD in maternal plasma: a 5 year experience in Cyprus.
abstract: BACKGROUND
  After the discovery that cell-free fetal DNA (cffDNA) is circulating in the maternal plasma of pregnant women, non-invasive prenatal diagnosis for fetal RhD in maternal plasma in RhD negative women at risk for haemolytic disease of the newborn (HDN) was clinically established and used by many laboratories. The objectives of this study are: (a) to assess the feasibility and report our experiences of the routine implementation of fetal RHD genotyping by analysis of cffDNA extracted from maternal plasma of RhD negative women at risk of HDN, and (b) to estimate the RhD phenotype frequencies, the RHD genotype frequencies and the RhD zygosity in the Cypriot population.
  
  
  METHODS
  cffDNA was extracted from maternal plasma of 73 RhD negative pregnant women. Real-Time Multiplex-PCR was used to amplify regions of RHD gene in exons 4, 5 and 10. RhD phenotypes were determined on 445 random samples using conventional agglutination slide test.
  
  
  RESULTS
  The fetus was predicted to be positive in 53 cases and negative in 18 cases. Two of cases were identified as D-variants, weak D type-1 and 11. The frequency of RhD negative homozygosity in the Cypriot population was estimated to be 7.2%, while the frequencies of RHD hemizygosity and RhD positive homozygosity was calculated to be 39.2 and 53.6%, respectively.
  
  
  CONCLUSIONS
  Fetal RHD genotyping can be accurately determined using cffDNA from maternal plasma. The implementation of the test has eliminated all use of unnecessary anti-D and reduced the total use of anti-D by 25.3% while achieving appropriate management of the RhD negative pregnancies.
authors.affiliation: Molecular Genetics Thalassaemia Department, The Cyprus Institute of Neurology and Genetics, 6 Internanional Airport Ave, Agios Dometios, 1683, Nicosia, Cyprus. thesalia@cing.ac.cy.
authors.forename: Thessalia
authors.initials: T
authors.lastname: Papasavva
authors.affiliation: International Blood Group Reference Laboratory, Bristol Institute for Transfusion Sciences, NHS Blood and Transport, North Bristol Park, Filton, Bristol, BS34 7QG, UK.
authors.forename: Pete
authors.initials: P
authors.lastname: Martin
authors.affiliation: Department of Transfusion Medicine, University Medical Center Göttingen, Robert-Koch-Straße 40, 37075, Göttingen, Germany.
authors.forename: Tobias J
authors.initials: TJ
authors.lastname: Legler
authors.affiliation: Zoodochou Pigis Clinic, 9 Antisthenous, Kapsalos, 3086, Limassol, Cyprus.
authors.forename: Marios
authors.initials: M
authors.lastname: Liasides
authors.affiliation: Mother and Child Medical Center, 9-11 Penelopis Delta Str., 1076, Nicosia, Cyprus.
authors.forename: George
authors.initials: G
authors.lastname: Anastasiou
authors.affiliation: Ultrasound Department, Makarios Hospital, 6 Koritsas Str., Strovolos, Nicosia, Cyprus.
authors.forename: Agathoklis
authors.initials: A
authors.lastname: Christofides
authors.affiliation: Apollonion Private Hospital, Lefkotheou 20, Strovolos, 2054, Nicosia, Cyprus.
authors.forename: Tasos
authors.initials: T
authors.lastname: Christodoulou
authors.affiliation: European Woman's Clinic, Vyzantiou 26, Strovolos, 2064, Nicosia, Cyprus.
authors.forename: Sotos
authors.initials: S
authors.lastname: Demetriou
authors.affiliation: Ygia Polyclinic Private Hospital, 21, Nafpliou str., 3305, Limassol, Cyprus.
authors.forename: Prokopis
authors.initials: P
authors.lastname: Kerimis
authors.affiliation: Iasis Hospital, 8 Voriou Ipirou str., 8069, Paphos, Cyprus.
authors.forename: Charis
authors.initials: C
authors.lastname: Kontos
authors.affiliation: G. Leontiades Clinic, 3 Apollonos Street, 6016, Larnaka, Cyprus.
authors.forename: George
authors.initials: G
authors.lastname: Leontiades
authors.affiliation: Mother and Child Medical Center, 9-11 Penelopis Delta Str., 1076, Nicosia, Cyprus.
authors.forename: Demetris
authors.initials: D
authors.lastname: Papapetrou
authors.affiliation: Patroklou Clinic, 14 Rubens Str., 3075, Limassol, Cyprus.
authors.forename: Telis
authors.initials: T
authors.lastname: Patroclos
authors.affiliation: Apollonion Private Hospital, Lefkotheou 20, Strovolos, 2054, Nicosia, Cyprus.
authors.forename: Marios
authors.initials: M
authors.lastname: Phylaktou
authors.affiliation: Ledra Obstetrics Gynecology Clinic, 19 Pindarou, Ayios Antonios, 1060, Nicosia, Cyprus.
authors.forename: Nikos
authors.initials: N
authors.lastname: Zottis
authors.affiliation: Molecular Genetics Thalassaemia Department, The Cyprus Institute of Neurology and Genetics, 6 Internanional Airport Ave, Agios Dometios, 1683, Nicosia, Cyprus.
authors.forename: Eleni
authors.initials: E
authors.lastname: Karitzie
authors.affiliation: Molecular Genetics Thalassaemia Department, The Cyprus Institute of Neurology and Genetics, 6 Internanional Airport Ave, Agios Dometios, 1683, Nicosia, Cyprus.
authors.forename: Eleni
authors.initials: E
authors.lastname: Pavlou
authors.affiliation: Molecular Genetics Thalassaemia Department, The Cyprus Institute of Neurology and Genetics, 6 Internanional Airport Ave, Agios Dometios, 1683, Nicosia, Cyprus.
authors.forename: Petros
authors.initials: P
authors.lastname: Kountouris
authors.affiliation: Sanquin Blood Supply, PO Box 9892, 1006 AN, Amsterdam, The Netherlands.
authors.forename: Barbera
authors.initials: B
authors.lastname: Veldhuisen
authors.affiliation: Sanquin Blood Supply, PO Box 9892, 1006 AN, Amsterdam, The Netherlands.
authors.forename: Ellen
authors.initials: E
authors.lastname: van der Schoot
authors.affiliation: Molecular Genetics Thalassaemia Department, The Cyprus Institute of Neurology and Genetics, 6 Internanional Airport Ave, Agios Dometios, 1683, Nicosia, Cyprus.
authors.forename: Marina
authors.initials: M
authors.lastname: Kleanthous
mesh_terms: D003535:Cyprus; D004899:Erythroblastosis, Fetal / Q000097:blood; D005260:Female; D005333:Fetus / Q000473:pathology*; D005787:Gene Frequency / Q000235:genetics; D006801:Humans; D008297:Male; D010641:Phenotype; D011247:Pregnancy; D011296:Prenatal Diagnosis / Q000379:methods*; D015995:Prevalence; D060888:Real-Time Polymerase Chain Reaction; D015203:Reproducibility of Results; D012204:Rh-Hr Blood-Group System / Q000097:blood* / Q000235:genetics
chemical_list: D012204:Rh-Hr Blood-Group System; C026410:Rho(D) antigen
keywords: Cell-free fetal DNA; Fetal RHD genotyping; NIPD; RhD frequency