Improving Fetal Fraction of Noninvasive Prenatal Screening Samples Collected in EDTA-Gel Tubes Using Gel Size Selection: A Head-To-Head Comparison of Methods.
Author(s): Daryabari SS, Giroux S, Caron A, Chau B, Langlois S, Rousseau F
Publication: J Mol Diagn, 2022, Vol. 24, Page 955-962
PubMed ID: 35820622 PubMed Review Paper? No
Purpose of Paper
The purpose of this paper was to compare the fraction of fetal cell-free DNA (cfDNA) in plasma from Streck tubes that were centrifuged after arrival and K2EDTA gel tubes that were centrifuged within 6 h of collection but shipped prior to plasma removal; effects of shipment delays and size selection were also investigated in plasma.
Conclusion of Paper
The average fraction of fetal DNA and the percentage of specimens with >4% fetal DNA were significantly higher in specimens collected in Streck tubes and processed within 5 days than for specimens collected in K2EDTA and shipped after centrifugation; and were nonsignificantly higher in K2EDTA specimens analyzed within 2 days compared to those that were not analyzed until 9 days. The fraction of fetal Y chromosome DNA was comparable among specimens collected in Streck and K2EDTA tubes, with the exception of the six K2EDTA specimens subjected to a shipment delay of 4-5 days during which they did not remain cold. When a cfDNA size selection step was added for K2EDTA tubes, the percentage of fetal DNA increased and sequence insert size decreased. Importantly, for specimens with a low fetal DNA fraction (including those due to high maternal body mass index, BMI), the authors report size selection was more effective at increasing the percentage of fetal DNA than repeat extraction, repeat library preparation, or deeper sequencing.
Studies
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Study Purpose
The purpose of this paper was to compare the fraction of fetal cfDNA in plasma from Streck tubes centrifuged after arrival and K2EDTA gel tubes that were centrifuged within 6 h but shipped prior to plasma removal; effects of shipment delays and size selection were also investigated in plasma. Blood was collected from sixty-one pregnant women (10-14 weeks) into K2EDTA gel tubes and Streck BCT. The K2EDTA tubes were centrifuged at 1800 x g for 10 min within 6 h of collection, stored at 4°C, and shipped overnight on cold packs. The Streck tubes were stored and shipped at room temperature. After arrival (2-9 days after collection), the K2EDTA tubes were decanted and plasma filtered through a 0.45 mmol/L HPF-Millex-PVDF Durapore filter. After arrival (2-5 days after collection), the Streck tubes were centrifuged at 1600 g for 20 min and the plasma filtered through a 0.45 mmol/L HPF-Millex-PVDF Durapore filter. cfDNA was isolated using the QIAamp Circulating Nucleic Acid Kit and quantified by the Qubit dsDNA HS assay. Libraries were prepared using the KAPA Hyper Prep Kit and pair-end sequenced on an Illumina NextSeq 550. Size selection of libraries was conducted using a LightBench instrument and 200/300 bp markers. The average fraction of fetal DNA was estimated using SeqFF. The percentage of fetal DNA was also calculated based on chromosome Y for the 25 women carrying male fetuses.
Summary of Findings:
The average fraction of fetal DNA for specimens collected in Streck tubes and processed within 5 days was significantly higher than those collected in K2EDTA tubes that were shipped after centrifugation within 2 days (8.6% versus 7.6%, P<0.001) or within 9 days of collection (8.6% versus 4%, P<0.001). Further, the percentage of specimens with less than 4% fetal DNA was lower when Streck tubes were used and analyzed within 5 days (11%) than when K2EDTA tubes were used and analyzed within 2 days (19%) or within 9 days (50%). While the percentage of fetal DNA and the percentage of specimens with >4% fetal DNA were higher in K2EDTA tubes that were analyzed within 2 days than those analyzed within 9 days, the difference was not significant. The fraction of fetal Y chromosome DNA was comparable from specimens collected in Streck and K2EDTA tubes, with the exception of six specimens that experienced a delay of 4-5 days and did not remain cold. When a size selection step was added for cfDNA from K2EDTA tubes, the percentage of fetal DNA increased for specimens analyzed within 2 days (7.6% to 13.3%) and within 9 days (4% to 10.4%), and no specimens had less than 4% fetal DNA. Importantly, for specimens with a low fetal DNA fraction (including those due to high maternal BMI), the authors report that size selection was more effective at increasing the percentage of fetal DNA than repeat extraction, repeat library preparation, or deeper sequencing. Library inserts from K2EDTA plasma and Streck plasma were as long as 200 bp, but after size selection of cfDNA from K2EDTA plasma the inserts were reduced to less than 160 bp.
Biospecimens
Preservative Types
- Streck/BCT
- None (Fresh)
Diagnoses:
- Pregnant
Platform:
Analyte Technology Platform DNA Next generation sequencing Pre-analytical Factors:
Classification Pre-analytical Factor Value(s) Biospecimen Acquisition Type of collection container/solution Streck tube
K2EDTA gel tube
Biospecimen Aliquots and Components Centrifugation Centrifugation delays investigated
Storage Storage duration <2 days
2-5 days
2-9 days