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Clinical experience and follow-up with large scale single-nucleotide polymorphism–based noninvasive prenatal aneuploidy testing

Open AccessPublished:August 08, 2014DOI:https://doi.org/10.1016/j.ajog.2014.08.006

      Objective

      We sought to report on laboratory and clinical experience following 6 months of clinical implementation of a single-nucleotide polymorphism–based noninvasive prenatal aneuploidy test in high- and low-risk women.

      Study Design

      All samples received from March through September 2013 and drawn ≥9 weeks’ gestation were included. Samples that passed quality control were analyzed for trisomy 21, trisomy 18, trisomy 13, and monosomy X. Results were reported as high or low risk for fetal aneuploidy for each interrogated chromosome. Relationships between fetal fraction and gestational age and maternal weight were analyzed. Follow-up on outcome was sought for a subset of high-risk cases. False-negative results were reported voluntarily by providers. Positive predictive value (PPV) was calculated from cases with an available prenatal or postnatal karyotype or clinical evaluation at birth.

      Results

      Samples were received from 31,030 patients, 30,705 met study criteria, and 28,739 passed quality-control metrics and received a report detailing aneuploidy risk. Fetal fraction correlated positively with gestational age, and negatively with maternal weight. In all, 507 patients received a high-risk result for any of the 4 tested conditions (324 trisomy 21, 82 trisomy 18, 41 trisomy 13, 61 monosomy X; including 1 double aneuploidy case). Within the 17,885 cases included in follow-up analysis, 356 were high risk, and outcome information revealed 184 (51.7%) true positives, 38 (10.7%) false positives, 19 (5.3%) with ultrasound findings suggestive of aneuploidy, 36 (10.1%) spontaneous abortions without karyotype confirmation, 22 (6.2%) terminations without karyotype confirmation, and 57 (16.0%) lost to follow-up. This yielded an 82.9% PPV for all aneuploidies, and a 90.9% PPV for trisomy 21. The overall PPV for women aged ≥35 years was similar to the PPV for women aged <35 years. Two patients were reported as false negatives.

      Conclusion

      The data from this large-scale report on clinical application of a commercially available noninvasive prenatal test suggest that the clinical performance of this single-nucleotide polymorphism–based noninvasive prenatal test in a mixed high- and low-risk population is consistent with performance in validation studies.

      Key words

      Since becoming clinically available in late 2011, cell-free DNA (cfDNA)-based noninvasive prenatal testing (NIPT) for fetal aneuploidy has seen an unprecedented rapid adoption into clinical care.
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      Validation of targeted sequencing of single-nucleotide polymorphisms for non-invasive prenatal detection of aneuploidy of chromosomes 13, 18, 21, X, and Y.
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      Here, laboratory and clinical experience of >31,000 women who received prenatal screening with a SNP-based NIPT is reported.

      Materials and Methods

      This is a retrospective analysis of prospectively collected data on 31,030 cases received for commercial testing from March through September 2013. This study received a notification of exempt determination from an institutional review board (Albert Einstein College of Medicine Institutional Review Board: no. 2014-3307). Samples were classified as out of specification and excluded in cases of gestational age <9 weeks, multiple gestation, donor egg pregnancy, surrogate carrier, missing patient information, sample received >6 days after collection, insufficient blood volume (<13 mL), wrong collection tube used, or if the sample was damaged.
      Analysis was performed for all samples on chromosomes 13, 18, 21, X, and Y, and included detection of trisomy 21, trisomy 18, trisomy 13, and monosomy X. All samples were processed and analyzed at Natera Inc’s Clinical Laboratory Improvement Act (CLIA)-certified and College of American Pathologists (CAP)-accredited laboratory (San Carlos, CA). Laboratory testing was performed as previously described using validated methodologies for cfDNA isolation, polymerase chain reaction amplification targeting 19,488 SNPs, high-throughput sequencing, and analysis with the next-generation aneuploidy test using SNPs (NATUS) algorithm.
      • Pergament E.
      • Cuckle H.
      • Zimmermann B.
      • et al.
      Single-nucleotide polymorphism-based noninvasive prenatal testing in a high-risk and low-risk cohort.
      • Nicolaides K.H.
      • Syngelaki A.
      • Gil M.
      • Atanasova V.
      • Markova D.
      Validation of targeted sequencing of single-nucleotide polymorphisms for non-invasive prenatal detection of aneuploidy of chromosomes 13, 18, 21, X, and Y.
      • Samango-Sprouse C.
      • Banjevic M.
      • Ryan A.
      • et al.
      SNP-based non-invasive prenatal testing detects sex chromosome aneuploidies with high accuracy.
      • Nicolaides K.H.
      • Syngelaki A.
      • Gil M.D.
      • Quezada M.S.
      • Zinevich Y.
      Prenatal detection of fetal triploidy from cell-free DNA testing in maternal blood.
      Samples were subject to a stringent set of quality-control metrics. A second blood draw (redraw) was requested if total input cfDNA, fetal cfDNA fraction, or signal-to-noise ratio did not meet quality metrics, or for poor fit of the data to the model. In cases of large regions (>25%) of loss of heterozygosity or suspected maternal or fetal mosaicism, redraw was not requested. Reports included a risk score for the 4 aneuploidies; when requested, reports included fetal sex. Risk scores were calculated by combining the maximum likelihood estimate generated by the NATUS algorithm with maternal and gestational age prior risks. All samples with a risk score ≥1/100 were reported as high risk for fetal aneuploidy and samples with risk scores <1/100 were considered low risk. For the purposes of this study, the high-risk results were further divided into a maximum-risk score of 99/100 or an intermediate-risk score of ≥1/100 and <99/100. The presence of >2 fetal haplotypes (indicative of either triploidy or multiple gestation) was reported only when the confidence was >99.9%. Additional sex chromosome aneuploidies (XXX, XXY, and XYY) were reported from June 2013. The following patient characteristics were requested for each sample: maternal date of birth, maternal weight, gestational age, and whether a paternal sample was included.
      Patients with available International Classification of Diseases, Ninth Revision (ICD-9) codes (Appendix; Supplementary Table 1) were categorized into 3 subcohorts: (1) “low risk” if aged <35 years and no aneuploidy-related high-risk codes; (2) “at risk” for fetal aneuploidy based solely on maternal age ≥35 years; or (3) “high risk” for fetal aneuploidy by ICD-9 code, regardless of maternal age. High-risk indications included positive screening tests, ultrasound anomalies, and relevant family history. Patients without reported ICD-9 codes were categorized by maternal age as low risk (<35 years) or high risk (≥35 years).
      Follow-up information on high-risk results was obtained by telephone and recorded in an internal database. Clinical follow-up was completed on June 14, 2014, at which time all pregnancies were completed. Two partner laboratories accounting for 38.1% of the total 31,030 cases were responsible for their own follow-up efforts and were excluded from outcome calculations. Providers were encouraged to share information about false-negative (FN) results. Samples were categorized as follows: (1) “true positive” (TP) included high-risk samples that were confirmed by prenatal or postnatal diagnostic testing, or based on clinical evaluation at birth; (2) “FP” included high-risk samples that were shown to be euploid by follow-up testing or based on clinical evaluation at birth; (3) “suggestive” included samples where prenatal ultrasound detected at least 1 structural anomaly and 1 soft sonographic marker consistent with NIPT findings, but karyotype confirmation was not obtained; (4) “pregnancy loss” where the patient experienced spontaneous abortion and karyotype confirmation was not obtained; (5) “termination” where the patient elected to end the pregnancy without karyotype confirmation; (6) “no follow-up” included samples where information was unavailable; and (7) “FN” included NIPT low-risk samples that were reported as aneuploid by the provider. When placental and fetal karyotypes were both available and determined to be discordant, NIPT findings were considered TP if they matched the fetal karyotype, and FP if they did not match the fetal karyotype. Pregnancies were considered mosaic when chromosome analysis revealed either placental or fetal mosaicism or there was discordance between placental and fetal karyotypes.
      Patient and sample characteristics were expressed as means, SD, medians, and ranges. Linear regression analysis was used to determine the relationship between fetal fraction and gestational age, between fetal fraction and maternal weight, and between fetal/maternal cfDNA and maternal weight; a reciprocal model was used when determining the relationship between fetal fraction and gestational age or maternal weight. For comparison of euploid and aneuploid calls, fetal fractions were expressed as multiples of the median (MoM) relative to low-risk calls weighted by week of gestation, and significance determined using a Mann-Whitney rank sum test. The 2 FN results were included in the appropriate aneuploid category, and FP calls were excluded from aneuploidy fetal fraction analyses. The benefit of a paternal sample on redraw rates and differences in aneuploidy incidence between the a priori risk groups were determined using a χ2 test. The Kruskal-Wallis 1-way analysis of variance on ranks test was used to evaluate maternal age and gestational age differences for the different risk groups. Positive predictive value (PPV) ([TP]/[TP + FP]) was calculated for cases with known cytogenetic analyses. SigmaPlot 12.5 (Systat Software, San Jose, CA) was used for all statistical analyses. P < .05 was considered statistically significant.

      Results

      Patients and samples

      Patient and sample characteristics for the 31,030 cases received during the study period are detailed in Table 1. Mean maternal age was 33.3 years, with 51.4% (15,952) aged ≥35 years at the estimated date of delivery. Mean gestational age was 14.0 weeks, with 64.5% (20,001) of samples drawn in first trimester and 33.8% (10,479) in the second trimester.
      Table 1Demographics of commercial cases
      DemographicWhole cohort, n = 31,030Follow-up cohort, n = 17,885
      Maternal age, y
      At estimated date of delivery
       Mean33.3 ± 6.033.7 ± 6.1
       Median35.035.0
       Range14.0–60.014.0–52.0
      Gestational age, wk
       Mean14.0 ± 4.414.5 ± 4.7
       Median12.613.0
       Range3.1–40.99.0–40.9
      As the follow-up cohort does not include any out-of-specification cases, or any cases that failed to receive a noninvasive prenatal testing result, minimum gestational age and fetal fraction are higher than in the whole cohort–however, mean values and SD are equivalent between the 2 cohorts
      Maternal weight, lb
      Analysis of maternal weight was limited to centers and laboratories that provided this information, and samples originating from United States to avoid inconsistent weight units.
       Mean158.4 ± 39.2157.2 ± 37.9
       Median149.0148.0
       Range83.0–425.083.0–385.0
      Fetal fraction, %
       Mean10.2 ± 4.510.8 ± 4.4
       Median9.610.1
       Range0.6–50.03.7–50.0
      As the follow-up cohort does not include any out-of-specification cases, or any cases that failed to receive a noninvasive prenatal testing result, minimum gestational age and fetal fraction are higher than in the whole cohort–however, mean values and SD are equivalent between the 2 cohorts
      Dar. Clinical performance of SNP-based NIPT. Am J Obstet Gynecol 2014.
      a At estimated date of delivery
      b As the follow-up cohort does not include any out-of-specification cases, or any cases that failed to receive a noninvasive prenatal testing result, minimum gestational age and fetal fraction are higher than in the whole cohort–however, mean values and SD are equivalent between the 2 cohorts
      c Analysis of maternal weight was limited to centers and laboratories that provided this information, and samples originating from United States to avoid inconsistent weight units.
      Figure 1 depicts the study flow chart. Samples from 325 (1.0%) patients were excluded as being outside of the specifications for testing (Supplementary Table 2) and 1966 samples failed quality-control metrics (Supplementary Table 3), mostly due to low fetal fraction, leaving 28,739 cases with NIPT results.
      Figure thumbnail gr1
      Figure 1Study flow chart
      OOS: see “Materials and Methods” section.
      OOS, out-of-specification; QC, quality control.
      Dar. Clinical performance of SNP-based NIPT. Am J Obstet Gynecol 2014.
      In 21,678 cases from clinics linking patient samples to a single case identification, 386 first draws did not meet requirements, thereby allowing analysis of redraw rates in 21,292 cases. A redraw was requested from 95.4% (1572/1648) of cases without a first draw result, 56.5% (888/1572) submitted a redraw, and 64.3% (571/888) of redraws were reported; 12 (2.1%) resolved redraws received a high-risk call. Redraw rates declined steadily over the reporting period (Figure 2); the most recent first sample redraw rates were 9.4% at 9 weeks’, and 5.4% at ≥10 weeks’ gestation. Around 30% of patients given the opportunity to submit a paternal sample chose to do so, and inclusion of a paternal sample was associated with a lower redraw rate, with a similar decline over the study period (Figure 2). This effect was more pronounced in women weighing >200 lb, where inclusion of a paternal sample reduced the redraw rate from 27.5% to 16.1% (P < .001). The average turn-around time was 9.2 calendar days (95% confidence interval [CI], 9.16–9.23 calendar days), but significant improvements over the study period led to an average turn-around time in the last month of 6.7 calendar days (95% CI, 6.68–6.76 calendar days).
      Figure thumbnail gr2
      Figure 2Father sample and clinical laboratory experience reduces redraw rate
      Decrease in redraw rates overall and for patients including a paternal sample during the reporting period (March through September 2013) for samples ≥10 weeks of gestation.
      Dar. Clinical performance of SNP-based NIPT. Am J Obstet Gynecol 2014.

      Fetal fractions

      The average fetal fraction was 10.2% (Table 1). Regression analysis, using the reciprocal of the independent variable (gestational age or maternal weight), revealed a positive correlation between fetal fraction and gestational age (r2 = 0.05, P < .001) (Figure 3, A), and a negative association between fetal fraction and maternal weight (r2 = 0.16, P < .001) (Figure 3, B). Furthermore, with increasing maternal weight, there was an increase in maternal cfDNA (P < .001) and a decrease in fetal cfDNA (P < .001) (Figure 4). Fetal fractions when stratified by aneuploidy were decreased for trisomy 13 (0.759 MoM, P < .001), trisomy 18 (0.919 MoM, P = .012), and monosomy X (0.835 MoM, P < .001), and increased for trisomy 21 (1.048 MoM, P = .018) samples.
      Figure thumbnail gr3
      Figure 3Effect of gestational age and maternal weight on fetal fraction
      Box plots depicting effects of A, gestational age and B, maternal weight on fetal fraction. Boxes indicate 75th (upper) and 25th (lower) quartiles, solid black line within box indicates median, capped whiskers indicate 90th (upper) and 10th (lower) percentiles, number in each grouping is indicated above 90th percentile whisker.
      Dar. Clinical performance of SNP-based NIPT. Am J Obstet Gynecol 2014.
      Figure thumbnail gr4
      Figure 4Increasing maternal weight increases maternal cfDNA and decreases fetal cfDNA
      Box plots depicting absolute levels of A, maternal and B, fetal cell-free DNA in maternal circulation as a function of maternal weight. Boxes indicate 75th (upper) and 25th (lower) quartiles, solid line within box indicates median, dashed line within box indicates mean, capped whiskers indicate 90th (upper) and 10th (lower) percentiles, diamonds indicate 95th (upper) and 5th (lower) percentiles.
      Dar. Clinical performance of SNP-based NIPT. Am J Obstet Gynecol 2014.

      NIPT results

      The combined rate of high-risk calls for all 4 indications was 1.77% (508/28,739); including 324 trisomy 21, 82 trisomy 18, 41 trisomy 13, and 61 monosomy X (Table 2). One sample was not assigned a risk score for chromosome 21 due to a maternal chromosome 21 partial duplication but was accurately identified as fetal trisomy 21 by the laboratory. Of 20,384 samples evaluated for additional sex chromosome aneuploidies, other than monosomy X, there were 14 (0.07%) identified: 6 XXX, 6 XXY, and 2 XYY. Fetal sex was reported in 24,522 cases. There were no reports of gender discordance from women receiving low-risk reports. For women receiving high-risk reports, confirmation of fetal sex was available for 109 cases, of which 108 (99.1%) were correct; the single discordant case was reported as high-risk for monosomy X (Supplementary Figure) but cytogenetic testing revealed a 46, XY fetus. Although cases with known multiple gestations were excluded, the NATUS algorithm identified 127 (0.4%) samples as having >2 fetal haplotypes, indicative of either unreported twins, vanishing twin, or triploidy.
      Table 2Number of fetal aneuploidy high-risk calls in reported commercial cases
      All cases, N = 28,739
      Total number of cases with reported result at ≥9 wk of gestation
      Trisomy 21Trisomy 18Trisomy 13Monosomy X
      Risk ≥99/100298
      Trisomy 21 and trisomy 18 totals include a single case of double-aneuploidy
      78
      Trisomy 21 and trisomy 18 totals include a single case of double-aneuploidy
      2653
      1/100 ≤ Risk <99/100254158
      Total324
      Trisomy 21 and trisomy 18 totals include a single case of double-aneuploidy
      Includes 1 case with a detected partial maternal chromosome 21 duplication, the fetus was determined to be high risk for trisomy 21 but the algorithm did not calculate a risk score.
      82
      Trisomy 21 and trisomy 18 totals include a single case of double-aneuploidy
      4161
      Prevalence, 1 in:88349697467
      Dar. Clinical performance of SNP-based NIPT. Am J Obstet Gynecol 2014.
      a Total number of cases with reported result at ≥9 wk of gestation
      b Trisomy 21 and trisomy 18 totals include a single case of double-aneuploidy
      c Includes 1 case with a detected partial maternal chromosome 21 duplication, the fetus was determined to be high risk for trisomy 21 but the algorithm did not calculate a risk score.
      ICD-9 codes were associated with 19.0% (5468/28,739) of women: 16.6% were low-risk, 44.1% were high-risk based only on advanced maternal age (≥35 years), and 39.3% had high-risk codes. As expected, the incidence of aneuploidy calls was smallest in the low-risk group (0.7%), followed by advanced maternal age women (1.6%), and largest in the high-risk group (3.4%) (Table 3). Results for the 23,271 samples without ICD-9 codes showed a similar difference in aneuploidy calls between women aged <35 years (1.0%, 117/11,629) and those aged ≥35 years (2.4%, 274/11,642).
      Table 3Aneuploidy calls in different a priori risk groups
      VariableCases with ICD-9 codes, n = 5468Cases without codes, n = 23,271
      Low risk, age <35 y (n = 909)AMA only, age ≥35 y (n = 2411)High risk, all ages (n = 2148)Low risk, age <35 y (n = 11,629)High risk, age ≥35 y (n = 11,642)
      Maternal age, y
      Mean ± SD, there was a significant difference between risk groups (P < .001) for both maternal age and gestational age, as determined by the Kruskal-Wallis 1-way analysis of variance on ranks test


      Median (range)
      28.2 ± 4.437.8 ± 2.431.3 ± 5.828.4 ± 4.537.9 ± 2.5
      29.0 (15.0–34.0)37.0 (35.0–48.0)32.0 (15.0–47.0)29.0 (14.0–34.0)37.0 (35.0–52.0)
      Gestational age, wk
      Mean ± SD, there was a significant difference between risk groups (P < .001) for both maternal age and gestational age, as determined by the Kruskal-Wallis 1-way analysis of variance on ranks test


      Median (range)
      14.1 ± 4.413.3 ± 3.515.8 ± 5.014.7 ± 4.913.4 ± 3.9
      12.4 (9.0–33.3)12.4 (9.0–38.1)14.4 (9.0–37.0)13.0 (9.0–38.0)12.1 (9.0–40.9)
      Euploid9032368207311,45711,293
      Trisomy 21227
      Trisomy 21 and trisomy 18 totals include single case of double-aneuploidy
      5057188
      Trisomy 1815
      Trisomy 21 and trisomy 18 totals include single case of double-aneuploidy
      132142
      Trisomy 131531121
      Monosomy X2262823
      Total aneuploids63872117274
      Monosomy X prevalence, %0.220.080.280.240.20
      Trisomy prevalence, %0.441.493.070.772.16
      Overall prevalence, %0.66
      Significant difference in aneuploidy call rate among 3 groups with ICD-9 codes (P < .001), as determined by χ2 test
      1.58
      Significant difference in aneuploidy call rate among 3 groups with ICD-9 codes (P < .001), as determined by χ2 test
      3.35
      Significant difference in aneuploidy call rate among 3 groups with ICD-9 codes (P < .001), as determined by χ2 test
      1.01
      Significant difference in aneuploidy call rate between 2 groups without ICD-9 codes (P < .001), as determined by χ2 test.
      2.35
      Significant difference in aneuploidy call rate between 2 groups without ICD-9 codes (P < .001), as determined by χ2 test.
      Women with ICD-9 codes were sorted into 3 risk populations based on ICD-9 codes and maternal age: low-risk women aged <35 y, women of AMA (aged ≥35 y) with no other high-risk codes, and high-risk women of any age. Women without ICD-9 codes were sorted into 2 risk populations based on maternal age: low-risk women aged <35 y and high-risk women of AMA.
      AMA, advanced maternal age; ICD-9, International Classification of Diseases, Ninth Revision.
      Dar. Clinical performance of SNP-based NIPT. Am J Obstet Gynecol 2014.
      a Mean ± SD, there was a significant difference between risk groups (P < .001) for both maternal age and gestational age, as determined by the Kruskal-Wallis 1-way analysis of variance on ranks test
      b Trisomy 21 and trisomy 18 totals include single case of double-aneuploidy
      c Significant difference in aneuploidy call rate among 3 groups with ICD-9 codes (P < .001), as determined by χ2 test
      d Significant difference in aneuploidy call rate between 2 groups without ICD-9 codes (P < .001), as determined by χ2 test.

      Follow-up of high-risk calls

      From 17,885 cases in the follow-up cohort, outcome information was sought for the 356 high-risk calls; 152 high-risk calls from the whole cohort described above were not contained within the follow-up cohort.
      Information regarding invasive testing uptake was available for 251/356 (70.5%) cases that received a high-risk result: 39.0% (139) elected invasive testing and 31.5% (112) declined invasive tests, and of the remaining 105 (29.5%), 39 had a spontaneous demise or elective termination. Within the 356 high-risk calls, there were in total 58 reported spontaneous abortions, including 16 cases categorized as TP, 2 FP, 4 with ultrasound findings suggestive of aneuploidy, and 36 with unconfirmed outcomes. There were 57 reported elective terminations, including 30 cases categorized as TP, 5 with ultrasound findings suggestive of aneuploidy, and 22 elective terminations with unconfirmed outcomes.
      At the conclusion of clinical follow-up, 62.4% (222/356) of high-risk calls had karyotype information or at-birth confirmation: 184 confirmed affected pregnancies (TP) and 38 unaffected pregnancies (FP) (Table 4). Eight cases showed placental or fetal mosaicism: 5 fetal mosaics (TP) were confirmed by amniocentesis (2 trisomy 21, 2 trisomy 18, 1 monosomy X), and 3 cases were considered FP because of confined placental mosaicism (CPM). Two CPM cases were high risk for trisomy 13 and were identified as mosaics by chorionic villus sampling (CVS), one was determined to be euploid by amniocentesis, and the other did not have a follow-up amniocentesis but ultrasound at 20 weeks was read as normal. In the third CPM case, at-birth testing revealed a 100% trisomy 18 placenta and a euploid child. Two FN results (both trisomy 21) were reported to the laboratory following amniocentesis due to other indications.
      Table 4Clinical follow-up findings
      N = 17,885
      Total number of cases with reported result at ≥9 wk of gestation from participating centers
      Trisomy 21Trisomy 18Trisomy 13Monosomy XTotal
      High-risk calls233
      Trisomy 21 and trisomy 18 totals include single double-aneuploidy case
      55
      Trisomy 21 and trisomy 18 totals include single double-aneuploidy case
      3038356
      Confirmed outcomes
       True positive140
      Includes 13 cases reported as trisomy 21 based on at-birth clinical evaluation
      2789184
       False positive14
      Includes 3 cases reported as normal based on at-birth clinical evaluation
      2
      Includes 1 confined placental mosaicism case
      13
      Includes 2 confined placental mosaicism cases (1 confirmed and 1 unconfirmed)
      Includes 1 case reported as normal based on at-birth clinical evaluation
      938
      Unconfirmed outcomes
       Suggestive
      Patients declined invasive testing but ultrasound findings were consistent with noninvasive prenatal testing findings (see “Materials and Methods” section)
      890219
       Pregnancy loss
      Patients experienced spontaneous abortion and did not obtain karyotype confirmation
      1863936
       Termination
      Patients chose to terminate pregnancy without diagnostic testing
      1430522
       No follow-up
      Follow-up information was not available
      3986
      One sample tested as high-risk (1/7.6) for fetal aneuploidy, analysis of second sample indicated that patient was at low-risk, follow-up information was not available.
      457
      Low-risk calls
      Confirmed outcomes
       False negative20002
      Dar. Clinical performance of SNP-based NIPT. Am J Obstet Gynecol 2014.
      a Total number of cases with reported result at ≥9 wk of gestation from participating centers
      b Trisomy 21 and trisomy 18 totals include single double-aneuploidy case
      c Includes 13 cases reported as trisomy 21 based on at-birth clinical evaluation
      d Includes 3 cases reported as normal based on at-birth clinical evaluation
      e Includes 1 confined placental mosaicism case
      f Includes 2 confined placental mosaicism cases (1 confirmed and 1 unconfirmed)
      g Includes 1 case reported as normal based on at-birth clinical evaluation
      h Patients declined invasive testing but ultrasound findings were consistent with noninvasive prenatal testing findings (see “Materials and Methods” section)
      i Patients experienced spontaneous abortion and did not obtain karyotype confirmation
      j Patients chose to terminate pregnancy without diagnostic testing
      k Follow-up information was not available
      l One sample tested as high-risk (1/7.6) for fetal aneuploidy, analysis of second sample indicated that patient was at low-risk, follow-up information was not available.
      For the sex chromosome aneuploidies XXX, XXY, and XYY, 7 of the 14 high-risk calls were within the follow-up cohort. Clinical follow-up revealed 4 cases with known outcomes: 2 TP (1 XXX, 1 XXY) and 2 FP (both XXX).
      Based on the cases with cytogenetic confirmation, women with an intermediate-risk score were more likely to have a FP result (19/24, 79.2%) than women with a maximum-risk score (19/198, 9.6%, P < .001). For the 36 cases that experienced spontaneous abortion and did not obtain karyotype confirmation, 33 (91.7%) had a maximum-risk score. All 22 patients who elected to terminate the pregnancy without confirmation had a maximal-risk score.

      Positive predictive value

      Based only on cases with cytogenetic diagnosis (Table 4), the PPV was 90.9% for trisomy 21 and 82.9% for all 4 cytogenetic abnormalities combined (Table 5). A theoretical PPV was also calculated under the 2 boundary conditions that all unconfirmed high-risk cases were either FP or TP (Table 5). This provided a range for the PPV of 60-94% for trisomy 21 and 52-89% for all abnormalities combined.
      Table 5Positive predictive values
      VariableTrisomy 21Trisomy 18Trisomy 13Monosomy XTotal
      Cytogenetically confirmed cases
       TP/(TP + FP) (PPV)140/154 (90.9%)27/29 (93.1%)8/21 (38.1%)9/18 (50.0%)184/222 (82.9%)
      All unconfirmed cases considered as FPs (lower bound)
       TP/(TP + FP) (PPV)140/233 (60.1%)27/55 (49.1%)8/30 (26.7%)9/38 (23.7%)184/356 (51.7%)
      All unconfirmed cases considered as TPs (upper bound)
       TP/(TP + FP) (PPV)219/233 (94.0%)53/55 (96.4%)17/30 (56.7%)29/38 (76.3%)318/356 (89.3%)
      PPV calculated as (TP)/(TP + FP). Data are presented for just those cases where there was cytogenetic or clinical confirmation of result; based on the extreme condition that all unconfirmed cases were FPs (lower bound) and the opposite condition that all unconfirmed results were TP (upper bound).
      FP, false positive; PPV, positive predictive value; TP, true positive.
      Dar. Clinical performance of SNP-based NIPT. Am J Obstet Gynecol 2014.
      Among women without ICD-9-coded indications, 63 women aged <35 years received high-risk calls, of which 39 (60.9%) had diagnostic testing and 34 were TP, a PPV of 87.2% (95% CI, 72.6–95.7%). Of 176 women ≥35 years with high-risk calls, 105 (59.7%) had confirmatory karyotyping and 87 were TP, a PPV of 82.9% (95% CI, 74.3–89.5%).

      Comment

      This report of initial clinical experience with this SNP-based NIPT in >31,000 pregnancies demonstrates that performance in clinical settings is consistent with validation studies.
      • Pergament E.
      • Cuckle H.
      • Zimmermann B.
      • et al.
      Single-nucleotide polymorphism-based noninvasive prenatal testing in a high-risk and low-risk cohort.
      • Nicolaides K.H.
      • Syngelaki A.
      • Gil M.
      • Atanasova V.
      • Markova D.
      Validation of targeted sequencing of single-nucleotide polymorphisms for non-invasive prenatal detection of aneuploidy of chromosomes 13, 18, 21, X, and Y.
      • Samango-Sprouse C.
      • Banjevic M.
      • Ryan A.
      • et al.
      SNP-based non-invasive prenatal testing detects sex chromosome aneuploidies with high accuracy.
      • Nicolaides K.H.
      • Syngelaki A.
      • Gil M.D.
      • Quezada M.S.
      • Zinevich Y.
      Prenatal detection of fetal triploidy from cell-free DNA testing in maternal blood.
      Using only cases confirmed through chromosome analysis or clinical evaluation at birth, the PPV in this mixed low- and high-risk population is 90.9% for trisomy 21 and 82.9% for all 4 aneuploidies, which is far better than current screening methods. Even under the highly conservative assumption that all unconfirmed high-risk cases are incorrect, this test still offers improved clinical performance over traditional screening.
      The main advantage of this study is the robust information it provides on clinical application of NIPT, which can contribute to, and improve, both test performance and counseling of patients. Fetal fraction, the main variable that affects redraw rates, is positively correlated with gestational age and negatively correlated with maternal weight, agreeing with previous studies.
      • Wang 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.
      • Poon L.C.
      • Musci T.
      • Song K.
      • Syngelaki A.
      • Nicolaides K.H.
      Maternal plasma cell-free fetal and maternal DNA at 11-13 weeks' gestation: relation to fetal and maternal characteristics and pregnancy outcomes.
      • Canick J.A.
      • Palomaki G.E.
      • Kloza E.M.
      • Lambert-Messerlian G.M.
      • Haddow J.E.
      The impact of maternal plasma DNA fetal fraction on next generation sequencing tests for common fetal aneuploidies.
      • Ashoor G.
      • Syngelaki A.
      • Poon L.C.
      • Rezende J.C.
      • Nicolaides K.H.
      Fetal fraction in maternal plasma cell-free DNA at 11-13 weeks' gestation: relation to maternal and fetal characteristics.
      There are 2 main clinical implications from these findings. First, adequate dating will lower the need for redraw, particularly at early gestational ages. Second, inclusion of a paternal blood sample significantly lowers redraw rates and should be offered to patients, particularly those >200 lb. Importantly, cases with extremely low fetal fraction, which typically do not resolve with redraw, may have an increased risk for fetal aneuploidy.
      • Pergament E.
      • Cuckle H.
      • Zimmermann B.
      • et al.
      Single-nucleotide polymorphism-based noninvasive prenatal testing in a high-risk and low-risk cohort.
      This is likely particularly important for maternal triploidy, which is associated with smaller placentas and lower fetal fractions,
      • Pergament E.
      • Cuckle H.
      • Zimmermann B.
      • et al.
      Single-nucleotide polymorphism-based noninvasive prenatal testing in a high-risk and low-risk cohort.
      • Nicolaides K.H.
      • Syngelaki A.
      • Gil M.D.
      • Quezada M.S.
      • Zinevich Y.
      Prenatal detection of fetal triploidy from cell-free DNA testing in maternal blood.
      and trisomy 13 and trisomy 18 pregnancies.
      In addition to determining the most likely ploidy state of a fetus, the NATUS algorithm also generates a chromosome-specific risk score, which is a measure of the probability of nonmosaic fetal aneuploidy. As expected, data showed that maximum-risk results are more likely to be TP than intermediate-risk results. Although a high-risk score appears to be more indicative of a TP result, individual numerical values should be interpreted cautiously. Regardless of the risk score, confirmatory studies must be offered to all women with positive results without exception. This is particularly important in light of the finding here that 6.2% of women with high-risk results chose to terminate the pregnancy without invasive test confirmation.
      Although referred to as fetal cfDNA, the primary source of cfDNA is placental trophoblast cells.
      • Taglauer E.S.
      • Wilkins-Haug L.
      • Bianchi D.W.
      Review: cell-free fetal DNA in the maternal circulation as an indication of placental health and disease.
      CPM, estimated to be present in 1-2% of 10- to 12-week gestations,
      • Choi H.
      • Lau T.K.
      • Jiang F.M.
      • et al.
      Fetal aneuploidy screening by maternal plasma DNA sequencing: “false positive” due to confined placental mosaicism.
      • Harrison K.J.
      • Barrett I.J.
      • Lomax B.L.
      • Kuchinka B.D.
      • Kalousek D.K.
      Detection of confined placental mosaicism in trisomy 18 conceptions using interphase cytogenetic analysis.
      impacts all NIPTs. Validation studies have typically excluded samples with fetal mosaicism or CPM. Yet, it is clear that when NIPT is performed in a clinical setting, the effect of mosaicism cannot be ignored, and its impact on FP and FN results should be addressed. In this cohort, 8/222 (3.6%) high-risk calls showed evidence of mosaicism. Two cases with CVS results that supported NIPT findings were later categorized as FPs because of CPM. Further, since most FPs in this cohort were determined by amniocentesis or at-birth testing without placental genetic analysis, there may be additional, undetected CPM cases within the FPs. From a retrospective analysis of CVS, Grati et al
      • Grati F.R.
      • Malvestiti F.
      • Ferreira J.C.
      • et al.
      The role of feto-placental mosaicism in false positive and false negative non-invasive prenatal screening (NIPS) results.
      estimated that the FP rate would be 0.08% for the 4 common aneuploidies. Our findings, combined with the known incidence of CPM-related FPs and FNs, further reinforce the need for adequate pretest counseling, as recommended by American Congress of Obstetrics and Gynecology (ACOG).
      American College of Obstetricians and Gynecologists
      Noninvasive prenatal testing for fetal aneuploidy. Committee opinion no. 545.
      Patients undergoing CVS following high-risk results with NIPT should be counseled that mosaic conditions can occur and later amniocentesis may be required.
      An unexpected finding in this study was that the PPV for women aged <35 years (87%) was similar to that of women aged ≥35 years (83%). This does not appear to be attributable to a bias in the referral of cases for karyotyping. Some women aged <35 years may have chosen NIPT because of ultrasound findings or positive results with traditional serum screening. However, the lower aneuploidy call incidence of 1.0% in women aged <35 years, vs 2.4% in women aged ≥35 years (Table 3), supports that these 2 groups of women do differ substantially with respect to aneuploidy incidence. The PPV was expected to be lower in low-risk women because the number of affected pregnancies would be lower but the number of FPs was predicted to be a constant proportion.
      • Benn P.
      • Cuckle H.
      • Pergament E.
      Non-invasive prenatal diagnosis for Down syndrome: the paradigm will shift, but slowly.
      The similar PPVs determined in both maternal age groups may indicate that FPs, like affected pregnancies, are also proportionately more common in older women; perhaps arising from trisomic conceptions that are rescued but express CPM. More data are needed to confirm this observation.
      Based on the current opinion statement from ACOG, NIPT is appropriate for use in high-risk patients.
      American College of Obstetricians and Gynecologists
      Noninvasive prenatal testing for fetal aneuploidy. Committee opinion no. 545.
      Nevertheless, the ability to detect aneuploidy with cfDNA depends on assay precision and fetal fraction, not on disease prevalence. Reported PPV in studies performed on mixed high- and low-risk populations, as well as the current study, far exceed current screening methodologies. Consistent with this, recent guidelines published by the American College of Medical Genetics and Genomics (ACMG) do not distinguish between high and low risk. Therefore, the transition of NIPT into a universal, first-line, aneuploidy screen should depend on the availability and affordability of NIPT, and not concerns about performance.
      In this cohort of women who were thought to have singleton pregnancies at the time of NIPT, 127 cases were identified as having >2 fetal haplotypes suggesting either triploidy or a previously undetected multifetal pregnancy or vanishing twin. The SNP-based NIPT methodology provided the opportunity to identify these cases, pursue further diagnostic avenues, and avoid FPs that can arise using alternative methodologies.
      • Futch T.
      • Spinosa J.
      • Bhatt S.
      • de Feo E.
      • Rava R.P.
      • Sehnert A.J.
      Initial clinical laboratory experience in noninvasive prenatal testing for fetal aneuploidy from maternal plasma DNA samples.
      The main limitation of this study is the incomplete follow-up data, particularly on low-risk patients, precluding precise calculation of sensitivity and specificity. While follow-up was not conducted on low-risk patients, given the clinical significance of a FN report, and based on our laboratory experience, it is likely that FNs would be voluntarily reported; there were 2 voluntarily reported FNs. However, the lack of comprehensive follow-up on all low-risk patients precluded determination of the negative predictive value. Nevertheless, it is important to note that strong performance characteristics were in keeping with prior validation studies,
      • Pergament E.
      • Cuckle H.
      • Zimmermann B.
      • et al.
      Single-nucleotide polymorphism-based noninvasive prenatal testing in a high-risk and low-risk cohort.
      • Nicolaides K.H.
      • Syngelaki A.
      • Gil M.
      • Atanasova V.
      • Markova D.
      Validation of targeted sequencing of single-nucleotide polymorphisms for non-invasive prenatal detection of aneuploidy of chromosomes 13, 18, 21, X, and Y.
      • Zimmermann B.
      • Hill M.
      • Gemelos G.
      • et al.
      Noninvasive prenatal aneuploidy testing of chromosomes 13, 18, 21, X, and Y, using targeted sequencing of polymorphic loci.
      even with the inclusion of mosaic samples. Follow-up of normal results remains an issue for all laboratories that wish to track results for quality assurance, and we support the ACMG recommendation for a national registry.
      • Gregg A.R.
      • Gross S.J.
      • Best R.G.
      • et al.
      ACMG statement on noninvasive prenatal screening for fetal aneuploidy.
      In conclusion, this is a large-scale report of clinical utilization of NIPT. Analysis of >31,000 samples from both low- and high-risk women supported that test performance of this NIPT method in a clinical setting mirrors the robust performance reported in validation studies.
      Clinical performance of SNP-based NIPT in a mixed high- and low-risk population is consistent with performance in validation studies. Similar PPVs were found in women aged <35 years and aged ≥35 years. The strength of the study is the robust information it provides on clinical application of NIPT. The primary limitation is the incomplete follow-up data, particularly on low-risk patients, precluding precise calculation of sensitivity and specificity.
      This study supports the use of NIPT as a first-line screening test for aneuploidy in all patients. Furthermore, it highlights the importance of, as well as provides data that can improve, counseling of patients. Finally, the results of this study raise the questions of how many FP results may be explained by CPM and how best to manage clinical care and diagnostic confirmation of high-risk NIPT results in light of potential CPM. The extent to which CPM may underlie NIPT FP results requires further investigation.

      Acknowledgments

      We would like to thank Steven Aldridge and Nia Sengupta for assistance with collecting and tracking follow-up information. We would also like to thank Dr Asim Siddiqui for critical review of the manuscript. N.S. and A.S. are employees of Natera Inc. S.A. was employed by Natera Inc during the study and initial follow-up period.

      Appendix

      Figure thumbnail fx1
      Supplementary Figure45,X/46,XY mosaicism may explain the single discordant fetal sex result
      Single-nucleotide polymorphism (SNP) data for single discordant fetal sex case are consistent with monosomy X fetus. Representative A, X-chromosome and B, Y-chromosome SNP plots from female (XX), male (XY), and monosomy X (45,X) fetuses are shown using samples with fetal fractions of around 10% (I) and 20% (II). X-axis of each SNP plot represents the position along the chromosome, and y-axis indicates allele ratio. A, Fetal SNP data are colored based on maternal genotype, with alleles arbitrarily labeled as A or B: where AA is blue, AB is green, and BB is red. When the maternal genotype is homozygous at a specific SNP location (red or blue dots), the presence of single X-chromosome (45,X fetus or XY fetus) can easily be distinguished from 2 X-chromosomes (XX fetus); 45,X fetus with single paternal X-chromosome has a different SNP profile to that shown, but is easily distinguished by the absence of maternal X-chromosome-derived SNPs in the fetus. B, Males are determined by the presence of Y-chromosome SNPs; as fetal fraction increases, Y-chromosome SNPs migrate further away from X-axis, but Y-chromosome SNPs remain detectable down to at least 4% fetal fraction. C, For the single discordant fetal sex case that had a fetal fraction of 10%, SNP data clearly indicate the presence of a single maternal X-chromosome, with no paternal X-chromosome or Y-chromosome detected, leading to the monosomy X result. Mosaicism, which is frequently seen in association with a 45,X cell line, is a possible explanation for this discordant result.
      NIPT, noninvasive prenatal testing.
      Dar. Clinical performance of SNP-based NIPT. Am J Obstet Gynecol 2014.
      Supplementary Table 1Prevalence of International Classification of Diseases, Ninth Revision codes in low-risk, high-risk, and advanced maternal age women
      ICD-9 codeDescriptionLR, nAMA, nHR, nCode type
      228.1Lymphangioma, any site102LR
      278Obesity, unspecified011LR
      293.84Anxiety disorder in conditions classified elsewhere100LR
      300Anxiety, dissociative and somatoform disorders–anxiety state unspecified0011LR
      305.03Alcohol abuse, in remission001LR
      305.1Tobacco use disorder (tobacco dependence)001LR
      306Physiological malfunction arising from mental factors–musculoskeletal001LR
      313.1Disturbance of emotions specific to childhood and adolescence–misery and unhappiness disorder100LR
      345Epilepsy and recurrent seizures001LR
      622.1Dysplasia of cervix600LR
      648.13Thyroid dysfunction–antepartum condition or complication–not delivered during current episode of care001LR
      649.13Obesity complicating pregnancy, childbirth, or puerperium–antepartum condition or complication–not delivered during current episode of care010LR
      649.43Epilepsy complicating pregnancy, childbirth, or puerperium (antepartum obstetric condition, not delivered during current episode of care)010LR
      655.53Suspected damage to fetus from drugs (antepartum condition or complication)121LR
      655.63Suspected damage to fetus from radiation010LR
      656.13Other known or suspected fetal and placental problems affecting management of mother–Rhesus isoimmunization100LR
      695.3Rosacea–acne001LR
      767.5Facial nerve injury–facial palsy002LR
      780.39Other convulsions010LR
      790.92Abnormal coagulation profile001LR
      795.79Other and unspecified nonspecific immunological findings (raised antibody titer, raised level of immunoglobulins)001LR
      V13.29Personal history of disease–other genital system and obstetric disorders001LR
      V13.63Personal history of congenital malformations of nervous system100LR
      V19.5Family history of skin condition111LR
      V22.0Supervision of normal first pregnancy21712LR
      V22.1Supervision of other normal pregnancy90524212133LR
      V22.2Pregnant state, incidental2886LR
      V23.41Pregnancy with history of preterm labor100LR
      V23.85Pregnancy resulting from assisted reproductive technology010LR
      V26.31Testing of female genetic disease carrier status46914761305LR
      V28.0Encounter for antenatal screening of mother–screening for chromosomal anomalies by amniocentesis021LR
      V28.1Screening for raised alpha-fetoprotein levels in amniotic fluid002LR
      V28.3Encounter for routine screening for malformation using ultrasonics201LR
      V28.6Encounter for antenatal screening of mother–screening for streptococcus B100LR
      V72.40Pregnancy examination or test–pregnancy unconfirmed010LR
      V72.42Pregnancy examination or test–positive result001LR
      V77.2Special screening for endocrine, nutritional, metabolic, and immunity disorders–malnutrition001LR
      V77.6Special screen for cystic fibrosis191919LR
      V77.7Special screen for other inborn errors of metabolism131414LR
      V78.2Special screen for sickle-cell disease131414LR
      V78.3Special screen for other hemoglobinopathies131414LR
      V82.9Unspecified condition100LR
      659.53AMA–first pregnancy29
      A small number of women assigned AMA codes but aged <35 y–and therefore not AMA–were included in low-risk cohort (n = 60).
      556116AMA
      659.6Elderly multigravida (unspecified as to episode of care or not applicable)011AMA
      659.63AMA–not first pregnancy33
      A small number of women assigned AMA codes but aged <35 y–and therefore not AMA–were included in low-risk cohort (n = 60).
      1489343AMA
      V23.82Supervision of other HR pregnancy, elderly primigravida0016AMA
      348Other conditions of brain001HR
      429.3Cardiomegaly (cardiac: dilatation, hypertrophy, Ventricular dilatation)001HR
      591Hydronephrosis001HR
      593.89Other specified disorders of kidney and ureter–other001HR
      606.9Male infertility, unspecified001HR
      628Infertility, female–associated with anovulation002HR
      628.8Infertility, female of unspecified origin002HR
      629.9Unspecified disorder of female genital organs001HR
      640Hemorrhage in early pregnancy, threatened abortion (unspecified as to episode of care or not applicable)002HR
      646.03Other complications of pregnancy, not elsewhere classified–papyraceous fetus (antepartum condition or complication)001HR
      646.3Recurrent pregnancy loss (unspecified as to episode of care or not applicable)001HR
      646.31Habitual aborter (for 646.3)001HR
      646.33Recurrent pregnancy loss (antepartum condition or complication not delivered during current episode of care)004HR
      655.03Central nervous system malformation in fetus–antepartum condition or complication0012HR
      655.13Chromosomal abnormality in fetus (antepartum condition or complication)00408HR
      655.23Hereditary disease in family possibly affecting fetus (antepartum condition or complication)0070HR
      655.8Other known or suspected fetal and placental problems affecting management of mother004HR
      655.83Other known or suspected fetal abnormality, not elsewhere classified–antepartum condition or complication00185HR
      655.9Known or suspected fetal abnormality affecting management of mother–unspecified (unspecified as to episode of care or not applicable)001HR
      655.93Known or suspected fetal abnormality affecting management of mother–unspecified (antepartum condition or complication)008HR
      656.43Intrauterine death (antepartum condition or complication)001HR
      656.53Poor fetal growth–antepartum condition or complication002HR
      658.03Oligohydramnios (antepartum condition or complication)002HR
      659.61Elderly multigravida (antepartum condition or complication)001HR
      659.73Abnormality in fetal heart rate or rhythm (antepartum condition or complication)001HR
      663.03Umbilical cord complication–prolapse of cord–presentation of cord (antepartum condition or complication)001HR
      663.83Other umbilical cord complications–velamentous insertion of umbilical cord004HR
      741Spina bifida with hydrocephalus–unspecified region001HR
      742.3Congenital hydrocephalus001HR
      742.4Other specified anomalies of brain003HR
      742.9Unspecified anomaly of brain, spinal cord, and nervous system001HR
      745.1Congenital anomalies–complete transposition of great vessels001HR
      745.4Ventricular septal defect001HR
      746.7Hypoplastic left heart syndrome001HR
      746.9Unspecified anomaly of heart–congenital001HR
      747.5Absence or hypoplasia of umbilical artery–single umbilical artery003HR
      747.89Other specified anomalies of circulatory system–other (aneurysm, congenital, specified site not elsewhere classified)001HR
      748.1Other anomalies of nose001HR
      753.29Obstructive defects of renal pelvis and ureter–other006HR
      754.7Other deformities of feet–talipes, unspecified001HR
      755.34Reduction deformities of lower limb–longitudinal deficiency, femoral, complete or partial (congenital absence of femur)001HR
      756.17Anomalies of spine–spina bifida occulta001HR
      758Down syndrome0018HR
      758.2Chromosomal anomalies–Edward syndrome0017HR
      758.5Other condition due to autosomal anomalies (fetal aneuploidy)006HR
      758.9Condition due to anomaly of unspecified chromosome001HR
      759.7Multiple congenital anomalies, so described002HR
      759.9Congenital anomaly, unspecified001HR
      764“Light for dates” without mention of fetal malnutrition001HR
      793.20Nonspecific (abnormal) findings on radiological and other examination of body structure–other intrathoracic organ0010HR
      793.60Nonspecific (abnormal) findings on radiological and other examination of body structure–abdominal area, including retroperitoneum001HR
      793.99Nonspecific (abnormal) findings on radiological and other examination of body structure–other (placental finding by x-ray or ultrasound method, radiological findings in skin and subcutaneous tissue)002HR
      796.5Abnormal/positive serum screening00548HR
      V13.69Personal history of other (corrected) congenital malformations001HR
      V18.4Family history of certain other specific conditions–intellectual disabilities001HR
      V18.9Family history of certain other specific conditions–genetic disease carrier003HR
      V19.8Family history of “other condition”00221HR
      V23.0Pregnancy with history of infertility00123HR
      V23.49Pregnancy with poor reproductive history (prior pregnancy with aneuploidy)0019HR
      V23.5Pregnancy with other poor reproductive history00123HR
      V23.81Supervision of other HR pregnancy0015HR
      V23.89Other HR pregnancy005HR
      V23.9Unspecified HR pregnancy006HR
      V26.89Other specified procreative management002HR
      V28.8Other specified antenatal screening0017HR
      V28.81Encounter for fetal anatomic survey001HR
      V28.89Other specified antenatal screening (CVS, genomic screening, nuchal translucency testing, proteomic screening)00441HR
      V28.9Unspecified antenatal screening00337HR
      All ICD-9 codes recorded in patients in this study were included in table.
      AMA, advanced maternal age; CVS, chorionic villus sampling; HR, high-risk; ICD-9, International Classification of Diseases, Ninth Revision; LR, low-risk.
      Dar. Clinical performance of SNP-based NIPT. Am J Obstet Gynecol 2014.
      a A small number of women assigned AMA codes but aged <35 y–and therefore not AMA–were included in low-risk cohort (n = 60).
      Supplementary Table 2Exclusion categories for out-of-specification samples
      Exclusion categoryCount
      Redraws accepted
       Insufficient serum/plasma127
       <9 wk of gestation
      Redraws are accepted once patient reaches 9 wks of gestation
      70
       Test cancelled45
       Sample collection date too old28
       Missing information11
       Sample damaged4
       Wrong tube4
       Other
      Includes uncommon exclusion reasons, such as hemolyzed blood samples and missing state-required waivers.
      26
      Redraws not requested
       Multiple gestation8
       Egg donor1
       Surrogate1
      Dar. Clinical performance of SNP-based NIPT. Am J Obstet Gynecol 2014.
      a Redraws are accepted once patient reaches 9 wks of gestation
      b Includes uncommon exclusion reasons, such as hemolyzed blood samples and missing state-required waivers.
      Supplementary Table 3Details of samples with failed quality metrics
      Exclusion categoryCount
      Redraws accepted
       Low fetal fraction1667
       Labchip QC failed48
       Contamination42
       Laboratory error34
       Unexplained bad model fit24
       Insufficient DNA17
       Uninformative single-nucleotide polymorphism pattern of unknown origin
      Unclear whether the uninformative single-nucleotide polymorphism pattern is maternal or fetal in origin.
      13
       Multiple sequencing failures9
      Redraws not requested
       Suspected egg donor/surrogate60
       Maternal loss of heterozygosity38
       Fetal loss of heterozygosity12
       Suspected maternal mosaicism1
       Suspected fetal mosaicism1
      QC, quality control.
      Dar. Clinical performance of SNP-based NIPT. Am J Obstet Gynecol 2014.
      a Unclear whether the uninformative single-nucleotide polymorphism pattern is maternal or fetal in origin.

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