The objective 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). We recruited 61 pregnant women at 10 to 14 weeks’ gestation undergoing NIPS. 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.,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. 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., 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., 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. 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. 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.,8Shi J. Zhang R. Li J. Zhang R. Size profile of cell-free DNA: a beacon guiding the practice and innovation of clinical testing. 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. 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.,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., 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., 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., 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. Different instruments were used to isolate these short fragments with electrophoresis using precast gels.14Leveraging 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?.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. 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., 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., 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. However, these specialized tubes are expensive, and the centrifugation and manual collection of plasma from these tubes is a labor-intensive low-throughput process. We previously reported that 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). 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, it would be convenient if a centrifuged gel tube could be sent over to a distant NIPS laboratory to be filtered without quality loss. The aims of this study are to determine if cfDNA in centrifuged EDTA-gel tube is protected from degradation for a long period of time compared with cell-stabilizing collection tubes and if libraries prepared from EDTA-gel tubes having travelled over long distances can be size selected to improve fetal fraction.

Herein, we report the results of a comparison among 61 samples collected in Vancouver (BC, Canada) in both K2EDTA tubes with gel and Streck tubes and shipped >4800 km to an NIPS testing laboratory in Quebec City (QC, Canada). We show that cfDNA collected from EDTA-gel conserves its complete integrity and that libraries constructed with this cfDNA can be rescued by gel electrophoresis, with results that surpassed those obtained from libraries prepared with cfDNA from Streck tube plasma without size selection.

Materials and MethodsEthics Statement

This study is part of a larger study named “Pegasus-2-Personalized Genomics for Prenatal Abnormalities Screening Using Maternal Blood: Toward First Tier Screening and Beyond” involving recruitment in different Canadian cities (NCT03831257). For the current study, pregnant women were exclusively recruited in Vancouver after consenting to participate to the study. The study was approved by UBC Research Ethics Board under number H18-02062 and by Comité d’éthique du CHU de Québec number MP-20-2019-4332.

Blood Collection

All singleton pregnant women were recruited between the months of August and October 2021 and were between 10 and 14 weeks of gestation. After consent, two 10-mL peripheral blood samples were collected in Streck tubes (catalog number 218997; Streck, La Vista, NE), and 8 mL was collected in one K2EDTA gel blood tube for molecular diagnostics (catalog number GR-455040; Greiner, Kremsmünster, Austria). EDTA-gel tubes were centrifuged at 1800 × g for 10 minutes within 6 hours of sample collection (as recommended by the manufacturer) and then kept at 4°C until shipped cold once a week to the Quebec City laboratory 4800 km away. Blood collected in Streck tubes was shipped at room temperature twice a week to the same laboratory. For both types of collection tubes, we used a priority overnight shipping.

Library PreparationStreck tubes were centrifuged for 20 minutes at 1600 × g, 2 to 5 days after blood collection. Plasma was collected carefully using a 1-mL pipetman or a transfer pipet. Plasma was filtered through a 0.45 μmol/L HPF-Millex-PVDF-Durapore from Millipore (Merck, MA) using a 5-mL syringe. The K2EDTA tube was quickly decanted into the 5-mL syringe cylinder and filtered using the same type of filter, as previously reported.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). EDTA-gel tubes spent 2 to 9 days between blood collection and filtration.

cfDNA was prepared from 2 to 5 mL plasma using the QIAamp Circulating Nucleic Acid kit (catalog number 55114; Qiagen, Hilden, Germany). DNA was recovered in 50 or 70 μL and quantified by fluorometry using the Qubit dsDNA HS Assay Kit (catalog number Q32851; Thermofisher, Waltham, MA). Volumes of 15 to 25 μL containing between 2 and 15 ng of cfDNA were used to prepare libraries using the KAPA Hyper Prep kit (catalog number 07962363001; Roche, Basel, Switzerland) following the manufacturer's instructions with some modifications. Volumes were reduced by half at every step. Kapa unique dual-indexed adapters (catalog number 08861919702; Roche) were used at 75 nmol/L final concentration. Ten cycles of amplification were performed, and a postamplification cleanup with 1× Kapa beads (catalog number 07983298001; Roche) was performed.

Libraries were quantified by fluorometry using the Qubit dsDNA HS Assay Kit, as above. Molarity was calculated using an average size of 325 bp. A pool of up to 25 equimolar libraries was prepared to sequence paired ends for 150 cycles on the Illumina NextSeq 550 instrument with a midoutput flow cell (catalog number 20024909; Illumina, San Diego, CA).

Pools of up to 25 equimolar libraries were size selected using a LightBench instrument from Yourgene Health Canada. A 25-μL sample plus loading buffer and 200/300 bp labeled markers were run together on a 3% precast gel (CG-10600-13). Software was programed to extract DNA ranging from 229 to 286 bp, which corresponds to insert sizes between approximately 100 and 160 bp. DNA recovered was quantified with Qubit, and mass was converted to molarity using an average size of 275 bp. Pools of up to 25 libraries were sequenced on midoutput flow cells, as above.

Reads AnalysisPaired-end sequencing was performed to allow calculation of fragments' length, but single-end data were used for the fetal fraction estimation with SeqFF.20Kim S.K. Hannum G. Geis J. Tynan J. Hogg G. Zhao C. Jensen T.J. Mazloom A.R. Oeth P. Ehrich M. van den Boom D. Deciu C. Determination of fetal DNA fraction from the plasma of pregnant women using sequence read counts. Reads were aligned to the hg19 human reference genome using BWA21Fast and accurate long-read alignment with Burrows-Wheeler transform. with maximal exact matches limited to one. The mapped reads kept for analysis were >35 bp and primary mapped with a minimal quality of 3. Duplicates were conserved. Each sample was sequenced with and without the gel size-selection protocol, and both sequencing results were analyzed the same way.ResultsPrevious work has shown that EDTA-gel tube was as good as Streck tube to collect blood and prepare cfDNA for NIPS without reduction of fetal fraction if blood samples were processed the same day.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). Herein, we tested the possibility of keeping the centrifuged EDTA-gel tube at 4°C and shipping it cold to the sequencing laboratory 4800 km away. Table 1 shows the cohort characteristics, and Table 2 shows the results.

Table 1Sample Characteristics

Table 2Fetal Fraction Measured with SeqFF, According to Treatment

Samples Collected in EDTA-Gel Tubes versus Streck Tubes

Using the standard procedure with Streck tube, all samples arrived at the laboratory within 5 days at room temperature. Seven women (11.4%) had a fetal fraction of <4% at first pass; and after a second analysis, five were rescued, and two (3.2%) remained at <4% and these had a body mass index (BMI) of ≥30 kg/m2.

With the EDTA-gel tubes, samples arrived in the laboratory once a week, so they were kept at 4°C until shipped with frozen packs with a priority overnight shipping with the chosen transporter. However, two shipments were delayed 4 and 5 days, and samples were no longer cold. It was found that the 14 samples included in those two shipments suffered from this delay and had a much lower fetal fraction than expected (Figure 1 and Figure 2A). The mean fetal fraction for those samples was 4% compared with a mean of 8.6% for the samples from the same women collected in Streck tubes (Figure 1). Furthermore, seven samples had a fetal fraction of n = 47) had a fetal fraction on average 1.0% lower than Streck tubes (a significant difference P Figure 1). Individually, most samples had a lower fetal fraction, but some were higher in EDTA-gel tubes (Figure 2A). There were nine samples at Figure 1). This reduced fetal fraction with time was not statistically significant given the small number of samples in each category.

Figure 1Mean fetal fraction from samples isolated from Streck tubes, EDTA-gel tubes, and EDTA-gel tubes with libraries size selected (SS) on 3% agarose gel, classified according to time spent between collection in Vancouver (BC, Canada) and filtration in Quebec City (QC, Canada) for the EDTA-gel tubes. All Streck tubes traveled for 1 or 2 days, and plasma was prepared within 5 days since the venipuncture. EDTA-gel tubes traveled also for 1 or 2 days, but they were kept at 4°C for longer time. Distribution was according to number of days before plasma stabilization. A total of 16 samples were kept 2 to 3 days between collection in Vancouver and filtration in Quebec City, 13 samples were kept 4 to 5 days, and 18 samples were kept 6 to 9 days. A total of 14 samples were delayed for 4 and 5 days in two shipping and arrived warm in Quebec City (T4-T5). The bar indicates the 95% CI.

Figure 2A: Individual fetal fraction estimated with SeqFF from samples isolated from Streck tubes, EDTA-gel tubes, and EDTA-gel tubes with libraries size selected (SS) on 3% agarose gel. B: Chromosome (Chr) Y read count ratio from 25 individual pregnancies with male fetus. Blue star indicates delayed samples. Samples are ordered according to increasing fetal fraction observed in Streck tubes.

Size Selection on Samples Previously Tested

Before using the samples collected in EDTA-gel tubes, the size selection was tested with samples previously tested in a clinical laboratory. Twenty samples with a mean fetal fraction of 9% and euploid fetus were analyzed. cfDNA was prepared from frozen leftover plasma samples prepared from Streck tubes (80%) or from EDTA-gel tubes stabilized the same day (20%). These libraries were run on a 3% gel electrophoresis using the Lightbench instrument from Yourgene Health Canada. Software was set to collect fragments from 229 to 286 bp. After sequencing and estimating the fetal fraction with SeqFF, the fetal fraction was increased for each sample, with a mean 14%. This group of samples was then chosen as reference group for further analysis.

A second group was tested with 1 trisomy 13 sample, 1 trisomy 21 sample, 2 trisomy 18 samples, and 21 plasma samples from pregnant women with a BMI >30 kg/m2 and/or low fetal fraction. The plasma was from Streck tubes (76%) or from EDTA-gel tubes (24%) processed the same day. The mean fetal fraction of the group was 6% before size selection and 12% after size selection. In addition, all aneuploidies were clearly identified, and their Z-score increased compared with libraries without size selection, thus confirming that the increase in fetal fraction is real.

Samples Collected in EDTA-Gel Tubes before and after Size SelectionAll libraries prepared from DNA isolated from plasma from EDTA-gel tubes were size selected the same way as those in the preliminary test. The material collected was sequenced, and the fetal fraction was evaluated with SeqFF as usual.20Kim S.K. Hannum G. Geis J. Tynan J. Hogg G. Zhao C. Jensen T.J. Mazloom A.R. Oeth P. Ehrich M. van den Boom D. Deciu C. Determination of fetal DNA fraction from the plasma of pregnant women using sequence read counts. Most size-selected libraries showed a significant increase in fetal fraction, and some 10% remained about the same (n = 6) or decreased slightly when fetal fraction was measured with SeqFF (Figure 2A). Most important, no sample had a fetal fraction below our reporting cutoff of 4% (Figure 2A). The minimum observed was 6.4% and was among samples with delayed shipment. The minimum was 8.2% among those received within 2 days of shipping.The Streck tubes having the lowest fetal fraction were mainly those from pregnant women with a high BMI (six of seven with BMI > 25 kg/m2). Interestingly, pregnancies with a low fetal fraction were those who benefited the most from size selection, even though they were collected in EDTA-gel tubes (Supplemental Figure S1). Indeed, it was observed that for some women with a high BMI, repeated extraction, new library preparation, or deeper resequencing too often did not succeed in improving fetal fraction but size selection did.Chromosome Y–Based Estimation of Fetal FractionPregnancies with male fetus (n = 25) allow evaluation of the fetal fraction with the ratio of Y chromosome fragments sequenced over all fragments (Y count/total count). A similar fetal fraction was observed between those collected in Streck tubes and those collected in EDTA-gel tubes, except for the six samples that arrived warm in delayed shipments (Figure 2B). After size selection, all samples showed an almost doubled quantity of detected Y reads (Figure 2B), further confirming the true nature of the size selection–induced enrichment in cfDNA fragments of fetal origin.Preservation of cfDNA Integrity in EDTA-Gel TubeGiven that paired-end sequencing was performed, it was possible to determine the size of the sequenced fragments. Figure 3 shows the typical size distribution of sequenced fragments before and after size selection as well as that of libraries obtained from a Streck tube (in red and only one for the sake of clarity, but a complete run is shown in Supplemental Figure S2). No difference is observed between Streck and EDTA-gel tubes without size selection. After size selection of libraries from EDTA-gel samples, all reads are 8Shi J. Zhang R. Li J. Zhang R. Size profile of cell-free DNA: a beacon guiding the practice and innovation of clinical testing.,22Sanchez C. Roch B. Mazard T. Blache P. Dache Z.A.A. Pastor B. Pisareva E. Tanos R. Thierry A.R. Circulating nuclear DNA structural features, origins, and complete size profile revealed by fragmentomics.

Figure 3Sizes of fragments sequenced were determined after mapping the paired-end reads from samples isolated from EDTA-gel tubes before and after size selection (SS). One sample is from Streck tube for comparison.

Compared with Streck tubes, EDTA-gel tubes showed no important loss in cfDNA recovered, and the library built with the DNA was as good (similar quantity). Also, the size distribution profile on gel was similar, with a peak around 167 bp (data not shown). The tube used and the selection on 3% agarose did not artifactually increase the fetal fraction by interfering with SeqFF counting because an increase in reads from chromosome Y in pregnancies with a male fetus was also observed (Figure 2B).Cost Reduction

In most countries, an EDTA-gel tube costs 10 times less than a Streck tube. Using a frozen gel pack is not expensive, although it can increase the parcel weight. Also, during winter, it is necessary to use an insulated container to protect samples taken in Streck tubes from cold, which also adds to the cost.

Size selection was performed on individual libraries as well as on a pool of libraries, and it was found much more convenient and economical to use pool. Up to 25 libraries can be pooled together to run on the gel, and the unused wells of the gel can be used later if kept in the refrigerator. There is no need to purify the collected fraction but only to quantify, dilute, and denature before loading a flow cell. It only adds 30 minutes to the whole process. The cost of the gel selection reagents is in fact only a fraction of the savings afforded by the swap between Streck and EDTA-gel blood collection tube. In addition, it reduces the number of failures, so it reduces the number of samples to reanalyze and thus the burden in the laboratory. This must be considered as a reduction in cost as well.

DiscussionStreck tubes or other similar cell-stabilizing tubes (Roche) are frequently recommended to collect blood in remote locations for NIPS. That is because it was shown to maintain the integrity of white blood cells, as well as protecting cell-free DNA from degradation over several days.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. Blood samples collected for NIPS in regular EDTA tubes yield suitable results up to 24 hours after collection.23Buysse K. Beulen L. Gomes I. Gilissen C. Keesmaat C. Janssen I.M. Derks-Willemen J.J. de Ligt J. Feenstra I. Bekker M.N. van Vugt J.M. Geurts van Kessel A. Vissers L.E. Faas B.H. Reliable noninvasive prenatal testing by massively parallel sequencing of circulating cell-free DNA from maternal plasma processed up to 24h after venipuncture. It was shown recently that the use of EDTA-gel tubes for NIPS samples was convenient to save time.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). Indeed, on low-speed centrifugation, a gel barrier is generated between blood cells and plasma, allowing researchers to decant quickly rather than aspirate slowly to avoid the buffy coat. In addition, it was shown that filtration could be substituted to high-speed second centrifugation, further simplifying NIPS sample processing without the need for an ultracentrifuge.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). However, EDTA-gel tubes have to be centrifuged within 6 hours of blood collection to properly separate the cells from the plasma. Streck tubes can be centrifuged up to 7 days after collection without a significant effect on cfDNA; however, such tubes are sensitive to exposure to an inadequate temperature, such as 4°C.24Hidestrand M. Stokowski R. Song K. Oliphant A. Deavers J. Goetsch M. Simpson P. Kuhlman R. Ames M. Mitchell M. Tomita-Mitchell A. Influence of temperature during transportation on cell-free DNA analysis.

In the present study performed in Canada, a country with a cold winter, it was found that exposure to low temperature can cause hemolysis, resulting in lower fetal fraction (personal observation). The results suggest that EDTA-gel tubes can be used even in distant locations from the NIPS laboratory when the sampling tube can be centrifuged close to the blood sampling site and kept at 4°C. However, relative to Streck tubes, a 1% lower mean fetal fraction with EDTA-gel tubes was observed; and most important, the number of samples with <4% fetal fraction was larger, which is not optimal. The loss appeared to be more important when the tubes were not kept at 4°C, as seen with sample shipments that were delayed. However, after size selection of smaller sequencing fragments by gel electrophoresis, a significant increase in fetal fraction for most samples is observed. After size selection, no sample was found to have a fetal fraction of <4%. Furthermore, with size selection, most EDTA-gel samples achieve a higher fetal fraction than with the regular process using Streck tubes. This is important because specialized tubes, such as Streck, are expensive, are made of fragile glass, and are sensitive to cold temperatures; and collection of NIPS-grade plasma is time-consuming for the NIPS laboratory staff. Furthermore, using cell-stabilizing tubes does not prevent having samples with low fetal fraction, particularly for pregnant women with a large BMI. Clearly, size selection can be performed on Streck tubes also to increase the fetal fraction, but the goal of the present study was to reduce the burden in the laboratory for plasma preparation and to reduce the overall cost.

Our study brings further information regarding the integrity of cfDNA from samples collected in EDTA-gel tubes. These results suggest that cfDNA integrity is maintained because no differences between fragment lengths of libraries after sequencing from either tube or differences on cfDNA size distribution profile on gel were noticed. Therefore, the reason for a lower fetal fraction in EDTA-gel tubes is the release of genomic DNA from maternal cells rather than degradation of fetal cfDNA.

One limitation of this study is that, within the population studied, no case of aneuploidy was identified. However, it is known that size selection improves the detection of aneuploidies of any chromosome, as shown with the preliminary work reported herein on known ploidies and by others.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. Also, the results from pregnancies with male fetuses indicate that chromosome Y fragments are not degraded and are enriched with size selection, so the same results should be expected from other chromosomes. In addition, the sequenced read size distribution profile showed no difference between sequenced reads originating from a Streck tube or an EDTA-gel tube before size selection. For the 61 pregnant women tested, the NIPS results were concordant between that obtained from Streck tube and the one from EDTA-gel tube size selected or not when the fetal fraction was sufficient.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 increased the fetal fraction substantially.