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Fetal RHD detection from circulating cell‐free fetal DNA in maternal plasma: validation of a diagnostic kit using automatic extraction and frozen DNA
Author(s) -
Londero D.,
Stampalija T.,
Bolzicco D.,
Castro Silva E.,
Candolini M.,
Cortivo C.,
Dreossi C.,
Fantasia I.,
Pecile V.,
De Angelis V.
Publication year - 2019
Publication title -
transfusion medicine
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.471
H-Index - 59
eISSN - 1365-3148
pISSN - 0958-7578
DOI - 10.1111/tme.12605
Subject(s) - genotyping , dna extraction , cell free fetal dna , polymerase chain reaction , concordance , cord blood , fetus , medicine , biology , andrology , prenatal diagnosis , pregnancy , genotype , immunology , genetics , gene
SUMMARY Objective This study aimed to validate non‐invasive RHD genotyping of cell‐free fetal DNA (cff‐DNA) using different DNA extraction methods and of fresh and frozen extracted cff‐DNA. Background Non‐invasive RHD genotyping of cff‐DNA predicts fetal RhD phenotype, allowing for the rational implementation of antenatal immunoprophylaxis and representing a big step forward in the management of RhD‐immunised women. Validation of a diagnostic method is mandatory before its clinical application. Methods RhD‐negative pregnant women were recruited at different gestational ages. The cff‐DNA extraction was carried out using manual and automatic methods in order to improve cff‐DNA yield and optimise the extraction. Fetal RHD genotyping was performed using a commercial real‐time polymerase chain reaction (PCR) kit, and the results were compared with postnatal serological RhD determination on cord blood. Results Overall, 133 plasma samples were examined for the validation process, and a total of 423 tests were performed. No differences have been observed between the two extraction methods or between fresh or frozen cff‐DNA regarding cff‐DNA stability and quality parameters. There was 100% concordance between fetal RHD genotyping of cff‐DNA and RhD phenotype on cord blood for both extraction methods on both fresh and frozen cff‐DNA. Conclusion Our study shows the reliability of automatic and manual cff‐DNA extraction methods and the possibility of freezing extracted cff‐DNA when performing RHD genotyping. This result might be relevant for improving laboratory work and organisation through the development of a standardised procedure for fetal RHD genotyping on cff‐DNA, laying the foundations for evidence‐based use of anti‐D Ig prophylaxis in RhD pregnant women.