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 language | English |
|---|---|
| Pages (from-to) | 34-38 |
| Number of pages | 5 |
| Journal | Blood Transfusion |
| Volume | 10 |
| Issue number | 1 |
| DOIs | |
| Publication status | Published - 2012 |
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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 journal › Article
}
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
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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 -