Improving Fetal Fraction of Noninvasive Prenatal Screening Samples Collected in EDTA-Gel Tubes Using Gel Size Selection

The aim of this study was to compare the use of EDTA-gel blood collection tubes with and without size selection to cell-stabilizing collection tubes for remote blood sampling for noninvasive prenatal screening (NIPS). Sixty-one pregnant women at 10 to 14 weeks’ gestation undergoing NIPS were recruited. Participants were phlebotomized with Streck and EDTA-gel tubes. EDTA-gel tubes were centrifuged before shipping. Libraries prepared from cell-free DNA (cfDNA) extracted from both types of tubes were sequenced on Illumina NextSeq 500, and fetal fraction was estimated using SeqFF. EDTA-gel tube libraries were size selected on agarose gel to eliminate cfDNA fragments >160 bp and resequenced. The main outcome measure was fetal fraction expressed as percentage of total cfDNA sequenced, calculated from sequence read counts (SeqFF). Streck tube samples showed an average 1% higher fetal fraction than centrifuged EDTA-gel tubes without size selection. This difference increased with temperature. When EDTA-gel samples' libraries were size selected, the mean fetal fraction increased from 7% to 13%, with no sample having fetal fraction <4%. Using EDTA-gel tubes reduces NIPS sampling cost and tube processing time in the laboratory. Also, using EDTA-gel tubes does not lead to cfDNA degradation. Size selection increases fetal fraction, reduces the number of test failures, increases NIPS clinical performance, and may be helpful in situations asking for a higher fetal fraction, such as twin pregnancies or screening for sub-chromosomal imbalances. The aim of this study was to compare the use of EDTA-gel blood collection tubes with and without size selection to cell-stabilizing collection tubes for remote blood sampling for noninvasive prenatal screening (NIPS). Sixty-one pregnant women at 10 to 14 weeks’ gestation undergoing NIPS were recruited. Participants were phlebotomized with Streck and EDTA-gel tubes. EDTA-gel tubes were centrifuged before shipping. Libraries prepared from cell-free DNA (cfDNA) extracted from both types of tubes were sequenced on Illumina NextSeq 500, and fetal fraction was estimated using SeqFF. EDTA-gel tube libraries were size selected on agarose gel to eliminate cfDNA fragments >160 bp and resequenced. The main outcome measure was fetal fraction expressed as percentage of total cfDNA sequenced, calculated from sequence read counts (SeqFF). Streck tube samples showed an average 1% higher fetal fraction than centrifuged EDTA-gel tubes without size selection. This difference increased with temperature. When EDTA-gel samples' libraries were size selected, the mean fetal fraction increased from 7% to 13%, with no sample having fetal fraction <4%. Using EDTA-gel tubes reduces NIPS sampling cost and tube processing time in the laboratory. Also, using EDTA-gel tubes does not lead to cfDNA degradation. Size selection increases fetal fraction, reduces the number of test failures, increases NIPS clinical performance, and may be helpful in situations asking for a higher fetal fraction, such as twin pregnancies or screening for sub-chromosomal imbalances. The introduction of noninvasive prenatal screening (NIPS) into clinical care in 20111Ehrich M. Deciu C. Zwiefelhofer T. Tynan J.A. Cagasan L. Tim R. Lu V. McCullough R. McCarthy E. Nygren A.O. Dean J. Tang L. Hutchison D. Lu T. Wang H. Angkachatchai V. Oeth P. Cantor C.R. Bombard A. van den Boom D. Noninvasive detection of fetal trisomy 21 by sequencing of DNA in maternal blood: a study in a clinical setting.Am J Obstet Gynecol. 2011; 204: 205.e1-205.e11Abstract Full Text Full Text PDF Scopus (387) Google Scholar,2Palomaki G.E. Kloza E.M. Lambert-Messerlian G.M. Haddow J.E. Neveux L.M. Ehrich M. van den Boom D. Bombard A.T. Deciu C. Grody W.W. Nelson S.F. Canick J.A. DNA sequencing of maternal plasma to detect Down syndrome: an international clinical validation study.Genet Med. 2011; 13: 913-920Abstract Full Text Full Text PDF PubMed Scopus (687) Google Scholar has provided pregnant women around the world with information about their risk for fetal aneuploidies. Whatever the platform used, most NIPS laboratories will report a failure rate between 1% and 5%.3Badeau M. Lindsay C. Blais J. Nshimyumukiza L. Takwoingi Y. Langlois S. Legare F. Giguere Y. Turgeon A.F. Witteman W. Rousseau F. Genomics-based non-invasive prenatal testing for detection of fetal chromosomal aneuploidy in pregnant women.Cochrane Database Syst Rev. 2017; 11: Cd011767PubMed Google Scholar, 4Welker N.C. Lee A.K. Kjolby R.A.S. Wan H.Y. Theilmann M.R. Jeon D. Goldberg J.D. Haas K.R. Muzzey D. Chu C.S. High-throughput fetal fraction amplification increases analytical performance of noninvasive prenatal screening.Genet Med. 2021; 23: 443-450Abstract Full Text Full Text PDF PubMed Scopus (17) Google Scholar, 5Pertile M.D. Flowers N. Vavrek D. Andrews D. Kalista T. Craig A. Deciu C. Duenwald S. Meier K. Bhatt S. Performance of a paired-end sequencing-based noninvasive prenatal screening test in the detection of genome-wide fetal chromosomal anomalies.Clin Chem. 2021; 67: 1210-1219Crossref PubMed Scopus (12) Google Scholar This failure is mainly due to a fetal fraction that is too low to make a confident call.6Blais J. Giroux S. Caron A. Clement V. Dionne-Laporte A. Jouan L. Gauthier J. MacLeod T. Moore R. Parker J. Swanson L. Zhao Y. Rouleau G. Karsan A. Langlois S. Rousseau F. Non-invasive prenatal aneuploidy testing: critical diagnostic performance parameters predict sample z-score values.Clin Biochem. 2018; 59: 69-77Crossref PubMed Scopus (5) Google Scholar At the molecular level, fetal cell-free DNA (cfDNA) can be distinguished from maternal cfDNA by the fact that fragments are shorter,7Fan H.C. Blumenfeld Y.J. Chitkara U. Hudgins L. Quake S.R. Analysis of the size distributions of fetal and maternal cell-free DNA by paired-end sequencing.Clin Chem. 2010; 56: 1279-1286Crossref PubMed Scopus (180) Google Scholar,8Shi J. Zhang R. Li J. Zhang R. Size profile of cell-free DNA: a beacon guiding the practice and innovation of clinical testing.Theranostics. 2020; 10: 4737-4748Crossref PubMed Scopus (27) Google Scholar differentially methylated, and enriched at specific locations.9Wang E. Batey A. Struble C. Musci T. Song K. Oliphant A. Gestational age and maternal weight effects on fetal cell-free DNA in maternal plasma.Prenat Diagn. 2013; 33: 662-666Crossref PubMed Scopus (284) Google Scholar Recently, several studies have reported that the use of high-concentration agarose gel electrophoresis to select the shortest fragments of a library prepared from cfDNA before sequencing substantially increases the fetal fraction.4Welker N.C. Lee A.K. Kjolby R.A.S. Wan H.Y. Theilmann M.R. Jeon D. Goldberg J.D. Haas K.R. Muzzey D. Chu C.S. High-throughput fetal fraction amplification increases analytical performance of noninvasive prenatal screening.Genet Med. 2021; 23: 443-450Abstract Full Text Full Text PDF PubMed Scopus (17) Google Scholar,10Qiao L. Zhang Q. Liang Y. Gao A. Ding Y. Zhao N. Zhang W. Li H. Lu Y. Wang T. Sequencing of short cfDNA fragments in NIPT improves fetal fraction with higher maternal BMI and early gestational age.Am J Transl Res. 2019; 11: 4450-4459PubMed Google Scholar, 11Xue Y. Zhao G. Qiao L. Lu J. Yu B. Wang T. Sequencing shorter cfDNA fragments decreases the false negative rate of non-invasive prenatal testing.Front Genet. 2020; 11: 280Crossref PubMed Scopus (8) Google Scholar, 12Qiao L. Mao J. Liu M. Liu Y. Song X. Tang H. Zhang Q. Li H. Lu Y. Liang Y. Wang T. Experimental factors are associated with fetal fraction in size selection noninvasive prenatal testing.Am J Transl Res. 2019; 11: 6370-6381PubMed Google Scholar, 13Hu P. Liang D. Chen Y. Lin Y. Qiao F. Li H. Wang T. Peng C. Luo D. Liu H. Xu Z. An enrichment method to increase cell-free fetal DNA fraction and significantly reduce false negatives and test failures for non-invasive prenatal screening: a feasibility study.J Transl Med. 2019; 17: 124Crossref PubMed Scopus (32) Google Scholar Different instruments were used to isolate these short fragments with electrophoresis using precast gels.14Underhill H.R. Leveraging the fragment length of circulating tumour DNA to improve molecular profiling of solid tumour malignancies with next-generation sequencing: a pathway to advanced non-invasive diagnostics in precision oncology?.Mol Diagn Ther. 2021; 25: 389-408Crossref PubMed Scopus (9) Google Scholar To obtain cfDNA, plasma must be prepared with caution to avoid contamination with genomic DNA from white blood cells. Blood samples need to be processed immediately after collection, and an additional high-speed (16,000 × g) centrifugation step for plasma preparation is required.15Barrett A.N. Zimmermann B.G. Wang D. Holloway A. Chitty L.S. Implementing prenatal diagnosis based on cell-free fetal DNA: accurate identification of factors affecting fetal DNA yield.PLoS One. 2011; 6: e25202Crossref PubMed Scopus (91) Google Scholar However, this high-speed centrifugation step is not always possible at the blood collection sites. The solution adopted by many phlebotomy sites is to use specialized blood sampling tubes designed to maintain the integrity of cfDNA, such as Streck tubes.16Norton S.E. Luna K.K. Lechner J.M. Qin J. Fernando M.R. A new blood collection device minimizes cellular DNA release during sample storage and shipping when compared to a standard device.J Clin Lab Anal. 2013; 27: 305-311Crossref PubMed Scopus (66) Google Scholar, 17Norton S.E. Lechner J.M. Williams T. Fernando M.R. A stabilizing reagent prevents cell-free DNA contamination by cellular DNA in plasma during blood sample storage and shipping as determined by digital PCR.Clin Biochem. 2013; 46: 1561-1565Crossref PubMed Scopus (135) Google Scholar, 18Wong D. Moturi S. Angkachatchai V. Mueller R. DeSantis G. van den Boom D. Ehrich M. Optimizing blood collection, transport and storage conditions for cell free DNA increases access to prenatal testing.Clin Biochem. 2013; 46: 1099-1104Crossref PubMed Scopus (107) Google Scholar However, these specialized tubes are expensive, and the centrifugation and manual collection of plasma from these tubes is a labor-intensive low-throughput process. The use of vacutainer K2EDTA with gel for molecular diagnostics is suitable for cfDNA analyses.19Giroux S. Badeau M. Jeuken J. Caron A. Girouard J. Rousseau F. Validation of a new protocol to collect and isolate plasma from pregnant women for noninvasive prenatal testing (NIPT).J Appl Lab Med. 2021; 6: 743-749Crossref PubMed Scopus (3) Google Scholar The use of these tubes followed by filtration was shown to be cost-effective and efficient to reduce time-consuming steps in the laboratory. However, despite the fact that most blood collection sites can centrifuge a blood tube, many sites are not equipped with facilities and staff to open the blood tube and filter the plasma located on top of the gel barrier after centrifugation. Therefore, be a centrifuged gel tube be to a NIPS to be without The of this study are to cfDNA in centrifuged EDTA-gel tube is from for a of time compared with cell-stabilizing collection tubes and libraries prepared from EDTA-gel tubes having can be size selected to improve fetal This the of a samples in in both K2EDTA tubes with gel and Streck tubes and to an NIPS testing in that cfDNA from EDTA-gel integrity and that libraries with this cfDNA can be by gel with that from libraries prepared with cfDNA from Streck tube plasma without size selection. This study is of a study for Using and in the pregnant women were in after to to the The study was by the of number and by number pregnant women were between the of and and were between 10 and 14 of blood samples were in Streck tubes number and was in K2EDTA gel blood tube for molecular diagnostics number EDTA-gel tubes were centrifuged at × for 10 of sample collection by the and at a to the Blood in Streck tubes was at a to the laboratory. shipping was used for both types of collection tubes. Streck tubes were centrifuged for at × to after blood was using a or a was a from using a The K2EDTA tube was into the and using the of as S. Badeau M. Jeuken J. Caron A. Girouard J. Rousseau F. Validation of a new protocol to collect and isolate plasma from pregnant women for noninvasive prenatal testing (NIPT).J Appl Lab Med. 2021; 6: 743-749Crossref PubMed Scopus (3) Google Scholar EDTA-gel tubes to between blood collection and cfDNA was prepared from to plasma using the number DNA was in or and by using the number of to between and of cfDNA were used to libraries using the number the with were by at number were used at of amplification were and a with number was Libraries were by using the as was calculated using an average size of A of to libraries was prepared to sequence for on the Illumina NextSeq with a cell number of to libraries were size selected using a from A sample and bp were on a precast gel was to DNA from to to between and DNA was with and was to using an average size of of to libraries were sequenced on as sequencing was to of were used for the fetal fraction with G. J. Tynan J. G. Zhao C. Oeth P. Ehrich M. van den Boom D. Deciu C. of fetal DNA fraction from the plasma of pregnant women using sequence read Diagn. PubMed Scopus Google Scholar were to the using H. R. and accurate with 2010; PubMed Scopus Google Scholar with to The for were bp and with a of were sample was sequenced with and without the gel and both sequencing were the has shown that EDTA-gel tube was as as Streck tube to collect blood and cfDNA for NIPS without of fetal fraction blood samples were processed the S. Badeau M. Jeuken J. Caron A. Girouard J. Rousseau F. Validation of a new protocol to collect and isolate plasma from pregnant women for noninvasive prenatal testing (NIPT).J Appl Lab Med. 2021; 6: 743-749Crossref PubMed Scopus (3) Google Scholar the of the centrifuged EDTA-gel tube at and shipping to the sequencing the and the with BMI in a new with to and tubes size size fraction with mean with fetal fraction in a new Using the standard with Streck tube, samples at the at temperature. women a fetal fraction of at and after a were and at and these a of the EDTA-gel samples in the a were at with with a shipping with the However, were and and samples were no was that the 14 samples in from this and a fetal fraction than and The mean fetal fraction for samples was compared with a mean of for the samples from the women in Streck tubes samples a fetal fraction of was the Streck tubes from the EDTA-gel tubes that a fetal fraction on average than Streck tubes difference most samples a fetal fraction, were higher in EDTA-gel tubes were samples at for in EDTA-gel tubes and samples with Streck tubes. was a between the time at were in the and the of fetal fraction This fetal fraction with time was not the number of samples in fetal fraction estimated with from samples from Streck EDTA-gel and EDTA-gel tubes with libraries size selected on agarose read from pregnancies with are to fetal fraction in Streck using the samples in EDTA-gel the size selection was with samples in a clinical laboratory. samples with a mean fetal fraction of and were cfDNA was prepared from plasma samples prepared from Streck tubes or from EDTA-gel tubes the libraries were on a gel electrophoresis using the from was to collect fragments from to sequencing and the fetal fraction with the fetal fraction was increased for with a mean This of samples was as for A was with trisomy trisomy 21 trisomy and 21 plasma samples from pregnant women with a BMI low fetal The plasma was from Streck tubes or from EDTA-gel tubes processed the The mean fetal fraction of the was before size selection and after size selection. were and their increased compared with libraries without size that the increase in fetal fraction is libraries prepared from DNA from plasma from EDTA-gel tubes were size selected the as in the The was sequenced, and the fetal fraction was with as G. J. Tynan J. G. Zhao C. Oeth P. Ehrich M. van den Boom D. Deciu C. of fetal DNA fraction from the plasma of pregnant women using sequence read Diagn. PubMed Scopus Google Scholar libraries showed a increase in fetal fraction, and about the or when fetal fraction was with no sample a fetal fraction of The was and was samples with The was of shipping. The Streck tubes having the fetal fraction were mainly from pregnant women with a BMI of with BMI pregnancies with a low fetal fraction were the most from size were in EDTA-gel tubes was that for women with a new library or too not in fetal fraction size selection with of the fetal fraction with the of fragments sequenced fragments A fetal fraction was between in Streck tubes and in EDTA-gel for the samples that in size samples showed an of the of the size enrichment in cfDNA fragments of fetal that paired-end sequencing was was possible to the size of the sequenced the size of sequenced fragments before and after size selection as as that of libraries from a Streck tube and for the of a is shown in difference is between Streck and EDTA-gel tubes without size selection. size selection of libraries from EDTA-gel are and can the as reported by J. Zhang R. Li J. Zhang R. Size profile of cell-free DNA: a beacon guiding the practice and innovation of clinical testing.Theranostics. 2020; 10: 4737-4748Crossref PubMed Scopus (27) Google C. B. T. P. B. E. R. DNA and size profile by 2021; 6: PubMed Scopus Google Scholar with Streck EDTA-gel tubes showed no in cfDNA and the library with the DNA was as Also, the size profile on gel was with a around bp not The tube used and the selection on agarose not increase the fetal fraction by with an increase in from in pregnancies with a was most an EDTA-gel tube 10 than a Streck Using a gel is not expensive, can increase the Also, during is to use an to samples in Streck tubes from to the Size selection was on libraries as as on a of and was and to use to libraries can be to on the and the of the gel can be used in the is no need to the fraction to and before a to the process. The cost of the gel selection is in fact a fraction of the by the between Streck and EDTA-gel blood collection reduces the number of failures, reduces the number of samples to and the in the laboratory. This must be as a in cost as Streck tubes or cell-stabilizing tubes are to collect blood in remote for is was shown to maintain the integrity of white blood as as cell-free DNA from several D. Moturi S. Angkachatchai V. Mueller R. DeSantis G. van den Boom D. Ehrich M. Optimizing blood collection, transport and storage conditions for cell free DNA increases access to prenatal testing.Clin Biochem. 2013; 46: 1099-1104Crossref PubMed Scopus (107) Google Scholar Blood samples for NIPS in tubes suitable to after K. L. C. C. J. van J.M. van A. noninvasive prenatal testing by sequencing of circulating cell-free DNA from maternal plasma processed to after Biochem. 2013; 46: PubMed Scopus Google Scholar was shown that the use of EDTA-gel tubes for NIPS samples was to S. Badeau M. Jeuken J. Caron A. Girouard J. Rousseau F. Validation of a new protocol to collect and isolate plasma from pregnant women for noninvasive prenatal testing (NIPT).J Appl Lab Med. 2021; 6: 743-749Crossref PubMed Scopus (3) Google Scholar on a gel barrier is between blood and to than to avoid the was shown that filtration be to high-speed NIPS sample processing without the need for an S. Badeau M. Jeuken J. Caron A. Girouard J. Rousseau F. Validation of a new protocol to collect and isolate plasma from pregnant women for noninvasive prenatal testing (NIPT).J Appl Lab Med. 2021; 6: 743-749Crossref PubMed Scopus (3) Google Scholar However, EDTA-gel tubes have to be centrifuged of blood collection to the from the Streck tubes can be centrifuged to after collection without a on such tubes are to to an such as M. R. Song K. Oliphant A. J. M. P. R. M. M. A. of during on cell-free DNA Diagn Ther. PubMed Scopus Google Scholar the study in a with a was that to low can in fetal fraction The that EDTA-gel tubes can be used in from the NIPS when the sampling tube can be centrifuged to the blood sampling and at However, to Streck a 1% mean fetal fraction with EDTA-gel tubes was and most the number of samples with fetal fraction was is not The to be when the tubes were not at as with sample that were However, after size selection of sequencing fragments by gel a increase in fetal fraction for most samples is size no sample was to have a fetal fraction of <4%. with size most EDTA-gel samples a higher fetal fraction than with the using Streck tubes. This is specialized such as are expensive, are of and are to and collection of plasma is time-consuming for the NIPS using cell-stabilizing tubes does not having samples with low fetal fraction, for pregnant women with a size selection can be on Streck tubes to increase the fetal fraction, the of the study was to reduce the in the for plasma preparation and to reduce the study information the integrity of cfDNA from samples in EDTA-gel tubes. that cfDNA integrity is no were between fragment of libraries after sequencing from tube or on cfDNA size profile on Therefore, the for a fetal fraction in EDTA-gel tubes is the release of genomic DNA from maternal than of fetal of this study is the no of aneuploidy was However, is that size selection improves the detection of of as shown with the reported on and by N.C. Lee A.K. Kjolby R.A.S. Wan H.Y. Theilmann M.R. Jeon D. Goldberg J.D. Haas K.R. Muzzey D. Chu C.S. High-throughput fetal fraction amplification increases analytical performance of noninvasive prenatal screening.Genet Med. 2021; 23: 443-450Abstract Full Text Full Text PDF PubMed Scopus (17) Google Scholar Also, the from pregnancies with that fragments are not and are enriched with size the be from the sequenced read size profile showed no difference between sequenced from a Streck tube or an EDTA-gel tube before size selection. the pregnant women the NIPS were between that from Streck tube and the from EDTA-gel tube size selected or not when the fetal fraction was studies have reported that the use of high-concentration agarose gel electrophoresis to select the shortest fragments of a library prepared from cfDNA before sequencing increased the fetal fraction N.C. Lee A.K. Kjolby R.A.S. Wan H.Y. Theilmann M.R. Jeon D. Goldberg J.D. Haas K.R. Muzzey D. Chu C.S. High-throughput fetal fraction amplification increases analytical performance of noninvasive prenatal screening.Genet Med. 2021; 23: 443-450Abstract Full Text Full Text PDF PubMed Scopus (17) Google Scholar,10Qiao L. Zhang Q. Liang Y. Gao A. Ding Y. Zhao N. Zhang W. Li H. Lu Y. Wang T. Sequencing of short cfDNA fragments in NIPT improves fetal fraction with higher maternal BMI and early gestational age.Am J Transl Res. 2019; 11: 4450-4459PubMed Google Scholar, 11Xue Y. Zhao G. Qiao L. Lu J. Yu B. Wang T. Sequencing shorter cfDNA fragments decreases the false negative rate of non-invasive prenatal testing.Front Genet. 2020; 11: 280Crossref PubMed Scopus (8) Google Scholar, 12Qiao L. Mao J. Liu M. Liu Y. Song X. Tang H. Zhang Q. Li H. Lu Y. Liang Y. Wang T. Experimental factors are associated with fetal fraction in size selection noninvasive prenatal testing.Am J Transl Res. 2019; 11: 6370-6381PubMed Google Scholar, 13Hu P. Liang D. Chen Y. Lin Y. Qiao F. Li H. Wang T. Peng C. Luo D. Liu H. Xu Z. An enrichment method to increase cell-free fetal DNA fraction and significantly reduce false negatives and test failures for non-invasive prenatal screening: a feasibility study.J Transl Med. 2019; 17: 124Crossref PubMed Scopus (32) Google Scholar The size selection to improve the detection of in the of H.R. Leveraging the fragment length of circulating tumour DNA to improve molecular profiling of solid tumour malignancies with next-generation sequencing: a pathway to advanced non-invasive diagnostics in precision oncology?.Mol Diagn Ther. 2021; 25: 389-408Crossref PubMed Scopus (9) Google S. J.M. A. H.R. size selection for short cell-free DNA fragments for circulating DNA and improves during One. 2018; 13: PubMed Scopus Google Scholar circulating cell-free DNA was shown to be shorter than cfDNA from P. J. and of plasma DNA in A. PubMed Scopus Google Scholar NIPS or cfDNA were used to isolate short fragments with electrophoresis using precast such as from to agarose from and agarose from H.R. Leveraging the fragment length of circulating tumour DNA to improve molecular profiling of solid tumour malignancies with next-generation sequencing: a pathway to advanced non-invasive diagnostics in precision oncology?.Mol Diagn Ther. 2021; 25: 389-408Crossref PubMed Scopus (9) Google Scholar this study that size selection improves the fetal fraction with the use of EDTA-gel are expensive, and to in the laboratory. However, for EDTA-gel tubes be at after centrifugation. that improve fetal fraction women by test failure are to improve test for twin pregnancies and the detection of sub-chromosomal imbalances. The study was of the by the for the of and of of and the length for this study was provided by Illumina with a NextSeq and by for a Chen for to the with fetal fraction from samples from Streck EDTA-gel and EDTA-gel tubes with libraries size selected on agarose to the pregnant The the with of fragments sequenced were determined after the paired-end from a with samples from Streck tubes.

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