Improving Fetal Fraction of EDTA-Gel NIPS Samples Using Gel-Based Size-Selection

  • Dr François Rousseau, MD, MSc, FRCPC, FCAHS

    FQR-S/MSSS/CHUQ Research Awardee in Health Technology Assessment and Evidence Based Laboratory Medicine, Professor, Department of Molecular Biology, Medical Biochemistry and Pathology at the Faculty of Medicine, Université Laval, Researcher, Centre de recherche du CHU de Québec


Objective : Evaluation of EDTA-gel blood collection tubes from remote locations for NIPS (Non-invasive prenatal testing).

Design: Paired comparisons

Population: 61 pregnant women from British Columbia aged 19 or over, 10 to 14 weeks gestation undergoing NIPS.

Methods: Participants were phlebotomized with both Streck and EDTA-gel tubes. EDTA-gel tubes were centrifuged prior to shipping. Libraries prepared from cfDNA extracted from both types of tubes were sequenced on Illumina NextSeq 500 and fetal fraction estimated using SeqFF. EDTA-gel tube libraries were further size-selected on agarose gel and resequenced. The three results from each patient were compared.
Main outcome measure: Fetal fraction (FF) expressed as a percentage of total cfDNA sequenced calculated from sequence read counts (SeqFF).

Results: Streck tubes samples showed an average 1% higher FF than centrifuged EDTA-gel tubes without size selection kept at 4°C after centrifugation and shipped on frozen packs. This difference increases with temperature. When EDTA-gel samples’ libraries were size-selected by gel electrophoresis to eliminate cfDNA fragments larger than 160bp, the mean FF increased from 7% to 13% with no samples having a FF < 4%.

Conclusions: EDTA-gel tubes reduces NIPS sampling cost and reduces tube processing time in the laboratory. In addition, after adding a size-selection step of the libraries, the FF is increased without degradation of cfDNA. The step reduces the number of test failures, increases NIPS clinical performance, and may be helpful in situations asking for a higher FF such as twin pregnancies or screening for sub-chromosomal imbalances.

Learning Objectives:

1. Discuss the importance and impact of fetal fraction on NIPS assay performance. 

2. Explain how gel-based size selection must be applied to enrich maternal sample for cfDNA fragments of fetal origin.


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