Implementing non-invasive RHD genotyping on cell-free foetal DNA from maternal plasma: The Pavia experience

Ilaria Sbarsi, Paola Isernia, Laura Montanari, Carla Badulli, Miryam Martinetti, Laura Salvaneschi

Research output: Contribution to journalArticle

Abstract

Background. The occurrence of cell-free foetal DNA in maternal circulation opens new possibilities in non-invasive antenatal diagnosis. Real-time polymerase chain reaction (PCR) analysis is an useful approach to foetal RhD blood group determination, thus being important in the prevention of haemolytic disease of foetus and new-born (HDFN). Study design and methods. Using real-time PCR assays we typed 20 samples of plasma, provided in a blinded fashion, from the International Reference Laboratory, two plasma samples sent by the "2010 Workshop on Molecular Blood Group Genotyping"; seven samples from D-negative mothers at the 16th week of gestation provided by our Hospital as prospective validation cases, and two plasma samples received from the "1 st Collaborative study establishing the sensitivity standard for non-invasive prenatal determination of foetal RHD genotype". To confirm the RHD typing of the seven prospective samples, PCR with sequence specific primers (PCR-SSP) was applied on genomic DNA from amniocytes (5 cases) and paternal peripheral blood (2 cases). Results. The results for the 31 investigated samples showed 100% concordance. Our measurable benefits were: confidence with a new technology, awareness of having gained the European standard level and increased self-assurance regarding the introduction of this typing technique in prenatal diagnostics. Discussion. These results demonstrate the feasibility and accuracy of our validation protocol. RHD typing on cell-free foetal DNA is a procedure which requires particular care and a great degree of expertise for diagnostic use. International collaborations are essential for monitoring the performance of laboratories in the absence of specific quality control programmes.

Original languageEnglish
Pages (from-to)34-38
Number of pages5
JournalBlood Transfusion
Volume10
Issue number1
DOIs
Publication statusPublished - 2012

Fingerprint

Mothers
Blood Group Antigens
Real-Time Polymerase Chain Reaction
DNA
Prenatal Diagnosis
Fetal Blood
Quality Control
Fetus
Genotype
Technology
Education
Pregnancy
Polymerase Chain Reaction

Keywords

  • Cell-free foetal DNA
  • HDFN
  • Non-invasive prenatal diagnosis
  • RHD genotyping
  • Validation protocol

ASJC Scopus subject areas

  • Hematology
  • Immunology and Allergy

Cite this

Implementing non-invasive RHD genotyping on cell-free foetal DNA from maternal plasma : The Pavia experience. / Sbarsi, Ilaria; Isernia, Paola; Montanari, Laura; Badulli, Carla; Martinetti, Miryam; Salvaneschi, Laura.

In: Blood Transfusion, Vol. 10, No. 1, 2012, p. 34-38.

Research output: Contribution to journalArticle

Sbarsi, Ilaria ; Isernia, Paola ; Montanari, Laura ; Badulli, Carla ; Martinetti, Miryam ; Salvaneschi, Laura. / Implementing non-invasive RHD genotyping on cell-free foetal DNA from maternal plasma : The Pavia experience. In: Blood Transfusion. 2012 ; Vol. 10, No. 1. pp. 34-38.
@article{99acf578ff9b4131b5cba16cf53318fb,
title = "Implementing non-invasive RHD genotyping on cell-free foetal DNA from maternal plasma: The Pavia experience",
abstract = "Background. The occurrence of cell-free foetal DNA in maternal circulation opens new possibilities in non-invasive antenatal diagnosis. Real-time polymerase chain reaction (PCR) analysis is an useful approach to foetal RhD blood group determination, thus being important in the prevention of haemolytic disease of foetus and new-born (HDFN). Study design and methods. Using real-time PCR assays we typed 20 samples of plasma, provided in a blinded fashion, from the International Reference Laboratory, two plasma samples sent by the {"}2010 Workshop on Molecular Blood Group Genotyping{"}; seven samples from D-negative mothers at the 16th week of gestation provided by our Hospital as prospective validation cases, and two plasma samples received from the {"}1 st Collaborative study establishing the sensitivity standard for non-invasive prenatal determination of foetal RHD genotype{"}. To confirm the RHD typing of the seven prospective samples, PCR with sequence specific primers (PCR-SSP) was applied on genomic DNA from amniocytes (5 cases) and paternal peripheral blood (2 cases). Results. The results for the 31 investigated samples showed 100{\%} concordance. Our measurable benefits were: confidence with a new technology, awareness of having gained the European standard level and increased self-assurance regarding the introduction of this typing technique in prenatal diagnostics. Discussion. These results demonstrate the feasibility and accuracy of our validation protocol. RHD typing on cell-free foetal DNA is a procedure which requires particular care and a great degree of expertise for diagnostic use. International collaborations are essential for monitoring the performance of laboratories in the absence of specific quality control programmes.",
keywords = "Cell-free foetal DNA, HDFN, Non-invasive prenatal diagnosis, RHD genotyping, Validation protocol",
author = "Ilaria Sbarsi and Paola Isernia and Laura Montanari and Carla Badulli and Miryam Martinetti and Laura Salvaneschi",
year = "2012",
doi = "10.2450/2011.0021-11",
language = "English",
volume = "10",
pages = "34--38",
journal = "Blood Transfusion",
issn = "1723-2007",
publisher = "Edizioni SIMTI",
number = "1",

}

TY - JOUR

T1 - Implementing non-invasive RHD genotyping on cell-free foetal DNA from maternal plasma

T2 - The Pavia experience

AU - Sbarsi, Ilaria

AU - Isernia, Paola

AU - Montanari, Laura

AU - Badulli, Carla

AU - Martinetti, Miryam

AU - Salvaneschi, Laura

PY - 2012

Y1 - 2012

N2 - Background. The occurrence of cell-free foetal DNA in maternal circulation opens new possibilities in non-invasive antenatal diagnosis. Real-time polymerase chain reaction (PCR) analysis is an useful approach to foetal RhD blood group determination, thus being important in the prevention of haemolytic disease of foetus and new-born (HDFN). Study design and methods. Using real-time PCR assays we typed 20 samples of plasma, provided in a blinded fashion, from the International Reference Laboratory, two plasma samples sent by the "2010 Workshop on Molecular Blood Group Genotyping"; seven samples from D-negative mothers at the 16th week of gestation provided by our Hospital as prospective validation cases, and two plasma samples received from the "1 st Collaborative study establishing the sensitivity standard for non-invasive prenatal determination of foetal RHD genotype". To confirm the RHD typing of the seven prospective samples, PCR with sequence specific primers (PCR-SSP) was applied on genomic DNA from amniocytes (5 cases) and paternal peripheral blood (2 cases). Results. The results for the 31 investigated samples showed 100% concordance. Our measurable benefits were: confidence with a new technology, awareness of having gained the European standard level and increased self-assurance regarding the introduction of this typing technique in prenatal diagnostics. Discussion. These results demonstrate the feasibility and accuracy of our validation protocol. RHD typing on cell-free foetal DNA is a procedure which requires particular care and a great degree of expertise for diagnostic use. International collaborations are essential for monitoring the performance of laboratories in the absence of specific quality control programmes.

AB - Background. The occurrence of cell-free foetal DNA in maternal circulation opens new possibilities in non-invasive antenatal diagnosis. Real-time polymerase chain reaction (PCR) analysis is an useful approach to foetal RhD blood group determination, thus being important in the prevention of haemolytic disease of foetus and new-born (HDFN). Study design and methods. Using real-time PCR assays we typed 20 samples of plasma, provided in a blinded fashion, from the International Reference Laboratory, two plasma samples sent by the "2010 Workshop on Molecular Blood Group Genotyping"; seven samples from D-negative mothers at the 16th week of gestation provided by our Hospital as prospective validation cases, and two plasma samples received from the "1 st Collaborative study establishing the sensitivity standard for non-invasive prenatal determination of foetal RHD genotype". To confirm the RHD typing of the seven prospective samples, PCR with sequence specific primers (PCR-SSP) was applied on genomic DNA from amniocytes (5 cases) and paternal peripheral blood (2 cases). Results. The results for the 31 investigated samples showed 100% concordance. Our measurable benefits were: confidence with a new technology, awareness of having gained the European standard level and increased self-assurance regarding the introduction of this typing technique in prenatal diagnostics. Discussion. These results demonstrate the feasibility and accuracy of our validation protocol. RHD typing on cell-free foetal DNA is a procedure which requires particular care and a great degree of expertise for diagnostic use. International collaborations are essential for monitoring the performance of laboratories in the absence of specific quality control programmes.

KW - Cell-free foetal DNA

KW - HDFN

KW - Non-invasive prenatal diagnosis

KW - RHD genotyping

KW - Validation protocol

UR - http://www.scopus.com/inward/record.url?scp=84855744935&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=84855744935&partnerID=8YFLogxK

U2 - 10.2450/2011.0021-11

DO - 10.2450/2011.0021-11

M3 - Article

C2 - 22153691

AN - SCOPUS:84855744935

VL - 10

SP - 34

EP - 38

JO - Blood Transfusion

JF - Blood Transfusion

SN - 1723-2007

IS - 1

ER -