Fetal RHD detection from circulating cell-free fetal DNA in maternal plasma: validation of a diagnostic kit using automatic extraction and frozen DNA.

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.

Combined effects of thinning and decline on fine root dynamics in a Quercus robur L. forest adjoining the Italian Pre-Alps.

Aims: Oak decline is a complex phenomenon, characterized by symptoms of canopy transparency, bark cracks and root biomass reduction. Root health status is one of the first stress indicators, and root turnover is a key process in plant adaptation to unfavourable conditions. In this study, the combined effects of decline and thinning were evaluated on fine root dynamics in an oak forest adjoining the Italian Pre-Alps by comparison of acute declining trees with non-declining trees, both with and without thinning treatment of surrounding trees. Methods: Dynamics of volumetric root length density (RLD V ) and tip density (RTD V ), root tip density per unit length of root (RTD L ), diameter, branching index (BI) and mycorrhizal colonization were monitored by soil coring over 2 years as possible descriptors of decline. Key Results: At the beginning of the experiment, the relationship between canopy transparency and root status was weak, declining trees having slightly lower RLD V (-20 %) and RTD V (-11 %). After a 1 year lag, during which the parameters were almost unaffected, BI and RLD V , together with tip density, tip vitality and mycorrhizal colonization, became the descriptors most representative of both decline class and thinning. Thinning of declining trees increased RLD V (+12 %) and RTD V (+32 %), but reduced tip mycorrhizal colonization and vitality over time compared with non-thinned trees, whereas the opposite occurred in healthy trees, together with a marked decrease in branching. After thinning, there was an initial reduction in the structure of the ectomycorrhizal community, although recovery occurred about 10 months later, regardless of decline severity. Conclusions: Decline causes losses of fine root length, and a moderate recovery can be achieved by thinning, allowing better soil exploration by oak roots. The close correlation between root vitality and mycorrhizal colonization and their deterioration after thinning indicates that decline does not benefit from reduced root competition, excluding the hypothesis of limited water and nutrient availability as a possible cause of the syndrome in this forest.