The analysis of cell-free DNA in maternal plasma allows noninvasive prenatal testing (NIPT) for fetal trisomies 21, 18, and 13; sex chromosome imbalances; and some microdeletion syndromes.1–4 Current NIPT methods count the relative number of cell-free DNA fragments present or look at polymorphic differences in maternal and fetal DNA components that can indicate the presence of an affected pregnancy. When the proportion of fetal DNA (fetal fraction) is below an acceptable level, some laboratories consider the test to be uninterpretable (“no result”). Other laboratories may not consistently measure or report fetal fraction or use a low fetal fraction cutoff in reporting.5 The ability to distinguish between affected and unaffected pregnancies is reduced when fetal fraction is low.6
Fetal fraction is inversely correlated with maternal weight7,8 and positively correlated with gestational age.8,9 Some maternal health conditions or medications may influence fetal fraction10–13 and low fetal fraction levels may also be associated with adverse outcomes.14–17 Trisomy 18 and 13 and digynic triploidy are more frequent when fetal fraction is very low,18–22 which is likely to be attributable to small placental volume.23 The association between low fetal fraction and fetal chromosome abnormalities places increased importance on the accurate measurement of fetal fraction and optimal management of women who do not receive a NIPT result as a result of low fetal fraction.
The goal of this study was to evaluate variables that might affect the chance that a redrawn NIPT test will be informative after a no result. Understanding a patient's chance of an informative redraw should be helpful in counseling and evaluating the optimal prenatal testing alternatives.
MATERIALS AND METHODS
We retrospectively reviewed all samples referred to a commercial laboratory (Natera, San Carlos, California) for NIPT from January 1, 2016, to October 1, 2016, and identified all cases in which an initial sample did not provide a measure of fetal copy number for chromosomes 21, 18, and 13 and sex chromosomes (no result cases). This laboratory used an approach for NIPT that looked for chromosome imbalance by evaluating multiple single-nucleotide polymorphisms (SNPs) and assessed whether the fetal contributions were consistent with imbalance.18,24 In all pregnancies in which the fetal fraction was less than 2.8%, analysis was not attempted and the test was classified as a no result. In some additional cases with fetal fraction between 2.8% and approximately 7%, the SNP patterns were not sufficiently informative to interpret the data with a high degree of confidence and these tests were also classified as no result. Both of these no result situations were considered to be due to low fetal fraction. We excluded cases in which the reason for a no result was not related to the test methodology, for example inadequate sample volume or test cancellation. Other test ineligibility reasons included rare cases in which redraw would not be expected to yield a result as a result of a lack of informative SNPs, for example where the uninformative pattern might be attributable to large regions of homozygosity. We also excluded cases in which there was no result for a single chromosome (Fig. 1). A full set of reasons for no result cases has been published elsewhere.9 When a redraw was recommended, this was provided without an additional fee.
There were 3,355 cases with a no result as a result of low fetal fraction and where a second maternal plasma sample was received. For these cases, data extracted from the Natera database included maternal weight, gestational age at the first and second sampling, days between sampling, initial fetal fraction, second fetal fraction, and whether the second sample provided a result. Cases in which the redraw occurred on the same day as the initial draw (two pregnancies) were excluded. To minimize the effect of gestational age errors, we also excluded cases in which there was greater than 7-day discordancy in the estimated date of delivery for the two draws (135 pregnancies) or when the redraw occurred after 28 days. The study cohort therefore comprised 2,959 pregnancies (Fig. 1).
Binary logistic analysis and χ2 tests were carried using SPSS with a P value of <.05 considered significant. Regression analyses were performed in an Excel spreadsheet. All data were entered by data entry professionals through a double data-entry process with validation by comparison.
The study was considered “exempt” by an investigational review board because the analysis was performed on fully deidentified data and thus was not considered to involve human participants (Ethical and Independent Review Services, Study Number: 17113-01).
Of the 242,607 initial samples, a result was obtained in 230,341 (94.94%), including 230,020 with a full result and 321 with a result on all but one chromosome (Fig. 1). Cases without a result, 12,266 (5.06%), included 1,539 (0.63%) in which the sample was not appropriate for an additional attempt and 10,727 (4.42%) in which a redraw was possible. The latter group included 8,605 cases (3.55% of all cases) in which a redraw was recommended as a result of low fetal fraction or low confidence results. Of all cases with an incomplete result, a redraw was recommended in 87.8% (11,048/12,587) of cases. The average gestational age at time of the initial blood draw for the cases with a redraw recommendation was 12.6 weeks. For all redraws, regardless of the reason for recommending a second sample, the overall rate of informative second samples was 2,835 of 4,018 (70.6%, 95% CI 69.1–72.0%).
Of the 8,605 cases with low fetal fraction or a low confidence result, a redraw sample was received in 3,355 cases (39%). Relative to those who did not, women who did submit a second sample had a somewhat higher fetal fraction (average 3.10% vs 2.98%), lower maternal weight (average 206 vs 213 pounds), and earlier gestational age (12.2 vs 12.9 weeks). After exclusions as described in “Methods,” there were 2,959 cases eligible for the study and this cohort was further explored to understand factors that affect the probability of an informative redraw (Table 1). Of the 2,959 cases, a result was obtained for the second sample in 1,861 (62.9%, 95% CI 61.1–64.6%).
Fetal fraction was generally higher at the time of redraw and this difference was approximately constant across the range of initial fetal fraction values (Appendix 1, available online at http://links.lww.com/AOG/B113). For the 2,959 cases with redraws, the average time between draws was 14 days and the average increase in fetal fraction between draws was 1.24% (median 0.80%). The distribution of change in fetal fraction was right-skewed with an extreme group of approximately 5% of cases showing a greater than 5% change in fetal fraction (Appendix 2, available online at http://links.lww.com/AOG/B113). The most extreme change in fetal fraction was 21.2% with the two plasma samples drawn 8 days apart.
A higher initial fetal fraction was associated with a higher probability of an informative redraw (Fig. 2). For example, for women with an initial fetal fraction in the 1.5–2% range, the average informative redraw rate was 27.8% (95% CI 22.1–34.3%), but when the fetal fraction was greater than 4% at the time of the initial test, the informative redraw rate was 86.5% (95% CI 83.5–89.1%).
To further explore the variables that affect an informative redraw, we next considered the effect of maternal weight (Table 2; Fig. 3). Generally, for any particular initial fetal fraction, informative redraw rates were inversely proportional to maternal weight; the importance of maternal weight was especially evident at low fetal fraction values. For example, for fetal fraction 2.5–3.0%, the informative redraw rate was 47.7% (95% CI 41.3–54.2%) for women greater than 240 pounds but 73% (95% CI 66.4–78.8%) for women less than 180 pounds. Nonetheless, maternal weight, by itself, was not necessarily an impediment for an informative redraw. For all three weight groups, the informative rate was greater than 60% when the initial fetal fraction was above 3%. For each of these three broadly defined maternal weight groups, and considering all fetal fraction values, the overall informative redraw rates were 75.2% (95% CI 72.4–77.9%), 62.5% (95% CI 59.4–65.6%), and 48.1% (95% CI 44.4–51.9%), respectively.
Fetal fraction does not change rapidly with gestational age in the late first trimester, when most NIPT is performed and, consistent with this, we observed only a weak association between the informative redraw rate and gestational age at the time of the initial sample (Appendix 3, available online at http://links.lww.com/AOG/B113). Although gestational age at the time of the initial draw appeared to be unimportant, a longer time interval between blood draws was associated with a greater increase in fetal fraction (Fig. 4) and higher informative redraw rates (Fig. 5). Performing the redraw 8 or 9 days after the initial draw resulted in substantially higher informative rates compared with 6 or 7 days. Further delay in redraw was associated with a more modest additional chance for an informative result.
Logistic regression analysis was used to model the binary outcomes, informative result or no result, using the observed initial fetal fraction, maternal weight, time between draws, and first attempt gestational age, as input variables (fetal fraction, maternal weight, and time between draws were each statistically significant; P<.001), but initial draw gestational age was not significant (Appendix 4, available online at http://links.lww.com/AOG/B113). The logistic regression potentially allows a computation of the informative redraw rate for any combination of the variables. Illustrative data are shown in Appendix 5, available online at http://links.lww.com/AOG/B113. Table 3 provides modeled expected redraw informative rates for women 75–300 pounds and fetal fractions 2.5–6.0% with 8 days between draws (ie, the earliest and most practical time that most redraws will occur).
In this study, we address a question of practical importance to women who do not receive a NIPT result as a result of a low fetal fraction or inconclusive data: What is the chance that a redraw will result in informative testing? We focused on a cohort of almost 3,000 NIPT cases with a redraw submitted for a SNP-based NIPT.
We show that the probability that a redraw will provide a result is dependent on the fetal fraction at the first draw, maternal weight, and time interval between draws, but is not strongly dependent on the gestational age at the time of the first sample. Although fetal fraction changes only slowly with gestational age in the late first trimester, most women will show a higher fetal fraction at the second draw. This can be explained by regression to the mean; women with an atypically low fetal fraction in the first sample are more likely to show an average fetal fraction when resampled. Interestingly, although cell-free DNA has a very rapid turnover,25 our observations indicate that patients with a low fetal fraction show steadily increasing levels of fetal fraction over an extended period after the initial draw (Fig. 4). Although fetal fraction increases slowly, by 8–9 days after the initial draw, there is sufficient cell-free DNA to expect a 55–60% chance for success. Additional delay in performing the redraw would result in only a modest further improvement (Fig. 5). In approximately 5% of cases, fetal fraction does show very large increases (greater than 5% fetal fraction). Other than maternal weight, gestational age, and karyotype, the factors that determine fetal fraction are currently poorly understood and the reason why some women show these large changes is unknown. Additional follow-up studies on women showing such changes are needed to determine whether these cases are associated with adverse outcomes.14–17,22
The univariate analyses presented here need to be interpreted cautiously because the results are based on cases in which a redraw was actually carried out and this may have favored those with highest chance for success based on all contributing variables. The logistic regression simultaneously considers all variables and bases the associated probability of an informative result on a continuous scale of data combinations. This is therefore the preferred, albeit computationally more difficult, method of estimating the probability. Although the data presented in this article and the calculations for an informative redraw are specific to the method currently offered by Natera Inc, the general approach should be applicable to all approaches to NIPT or for subsets of women for whom redraw could be a considered an acceptable, practical, and safe option.
It is important to recognize the association between test failure resulting from low fetal fraction and increased risk for trisomy 18 and 13 and digynic triploidy.18–22 The American College of Obstetricians and Gynecologists and the American College of Medical Genetics and Genomics suggest somewhat different approaches to the management of women who do not receive a result from the first NIPT test. The American College of Obstetricians and Gynecologists states that women whose initial test results are not reported should receive further genetic counseling and be offered comprehensive ultrasound evaluation and diagnostic testing because of an increased risk of aneuploidy, but does not preclude repeat NIPT as an option.26 Conversely, the American College of Medical Genetics and Genomics states that a repeat blood draw is not appropriate and that diagnostic testing should be offered.27 However, given the relationship between maternal weight and fetal fraction, not all women with a no result from the first NIPT attempt would be at the same risk for a fetus affected with trisomy 13 and 18 or triploidy.
In summary, we suggest that estimating the chance of an informative redraw could aid in the counseling of women who are faced with the choice of reattempting NIPT, conventional screening, or an invasive diagnostic test.
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