Introduction
Trisomy 21 (T21, Down syndrome), trisomy 18 (T18, Edwards syndrome), and trisomy 13 (T13, Patau syndrome) are the most common autosomal aneuploidies that cause mental retardation and serious congenital defects of newborns [
1‐
3], the prevalence per 10,000 live births is 12.3–17.26, 3.43–4.08 and 1.49–1.68 for T21, T18 and T13, respectively [
4‐
6], and the number would be higher if counting the fetal loss and pregnancy termination after prenatal diagnosis. Among the life birth of infants with T18 or T13, about 50% of them would not survive the first week and only about 10% of them would survive the first year [
3]. Whereas 88.9% in 1983 and 87.5% in 2006 of live birth infants with T21 would live to at least 15 and 25 years, respectively [
7], and many of them would live to 50 years or older [
8]. Even though with a relatively longer life expectancy, other than intellectual disability, individuals with Down Syndrome almost all suffer one or more long term health problems through their lifespan, such as cardiac complications, pulmonary hypertension, hematologic and oncologic disorders, neurodevelopmental disorders, respiratory complications, dysphagia, and others[
1,
9]. Recent publications investigated the impacts of COVID-19 disease on individuals with Down syndrome and concluded that the above health conditions might make these individuals at higher risk of SARS-CoV-2 infection or more severe clinical symptoms than their counterparts without Down syndrome [
10‐
12]. Currently, there is no cure for Down’s syndrome, so prenatal detection would provide affected families the options of preparing the birth of a child with Down’s syndrome or terminating the current pregnancy. Conventionally, screen of the fetal aneuploidies is normally done in the first and second trimesters by testing the maternal serum serological makers, typically including pregnancy associated plasma protein A (PAPP-A), human chorionic gonadotropin (hCG), alpha-fetoprotein (AFP), unconjugated estriol (uE3), and Inhibin A. These markers are normally grouped as double, triple, or quadruple assays with or without in combinations with maternal age. These serum biochemical tests (SBT) showed a positive detection rate of 49–76% with 5% false positive rate[
13]. The high risk pregnancies identified by the SBT are then referred to chorionic villus sampling (CVS) or amniocentesis (AC) tests that are defined as the gold stand for final diagnosis of the fetal aneuploidies, however, these two invasive procedures are time consuming and may also have up to 1% miscarriage rate[
14‐
16]. Instead of quantitatively and directly measuring the chromosomal dosages related to T21, T13 and T13, the SBT targets surrogate serological markers and indirectly estimates the risks or probabilities if a pregnancy would carry a fetus with aneuploidy. Unavoidably, in addition to a relative low sensitivity, the SBT also have a high false positive rate. In fact, about 95% of the “high risk” pregnancies identified by the SBT were false positive and would unnecessarily undergo through the expensive and stressful invasive CVS or AC test, as well as taking a risk of miscarriage [
17,
18].
Since the discovery of cell free fetal DNA (cffDNA) in maternal plasma in 1997[
19], several nucleic acid detection technologies (NADT) have, in proof-of-concepts, applied to the noninvasive prenatal test (NIPT) of aneuploidies, such as next generation sequencing (NGS) [
20,
21], DNA methylation [
22], Matrix-assisted laser desorption/ionization-time of flight mass spectrometry [
23], microarray [
24], and digital PCR [
25]. So far, the NGS is the most successful and recognized NADT used for prenatal screens of fetal aneuploidies, recent mete-analyses indicated that the pooled sensitivity for T21, T18, and T13 were in a range of 99.3–99.4%, 97.4–97.7%, and 90.6–97.5%, respectively, while the pooled specificity was 99.9% for these three trisomies [
26‐
29]. Since its clinical implementations, more affected pregnancies have been identified, meanwhile the number of CVS and AC procedures have dramatically dropped. However, the high overall costs and operational complexities of the NGS-NIPT might hinder its implementation to a wide-spread clinical routine practice. Even though, the NGS-NIPT has been recommended in many counties as the first-tier screen method for fetal aneuploidies screen, majority of pregnant women cannot afford to choose this test because it is currently not universally covered by healthcare systems or private insurances [
30,
31]. To effectively balance the benefits and higher cost regarding the NGS-NIPT, many countries select the contingent strategy (CS) that uses the SBT as a primary screen tool and use the NGS-NIPT as a confirmation test for the high risk pregnancies identified by the SBT. The CS showed a better cost-effectiveness when compared to the SBT only strategy and NGS-NIPT as the first-tier test [
32‐
35]. The main advantage of the CS is its ability to remarkably reduce the unnecessary invasive procedures, but its positive detection rate would in principle not be much better than the SBT, since all high risk pregnancies are still determined by the SBT at the forefront of this strategy [
35]. However, if the SBT can be replaced by a NADT assay with a higher sensitivity similar to the NGS-NIPT and a lower price close to the SBT, the upgraded CS would currently be a preferred solution to screen all pregnancies. Digital PCR (dPCR) is one of the most suitable NADT platforms for replacing the SBT due to its incomparable capability in absolute quantification of nucleic acids [
36]. In the dPCR process, the PCR reaction mix containing targets of interests is randomly distributed into tens of thousands independent partitions and each partition contains one (or few) or no target molecule. After PCR, the proportion of positive partitions is applied to accurately quantify the target concentration using Poisson’s statistics [
36‐
38]. There are quite a few publications demonstrated the usefulness of dPCR technology in detection of fetal T21 from variety of angles, some used the earlier versions of dPCR platforms or invasive CVS/AS samples to prove the concept [
25,
39,
40], while others evaluated the feasibilities in detections of fetal T21 and T18 in maternal plasma samples [
41‐
44].
Here we describe the performances of a multiplex dPCR-NIPT technique for simultaneous detections of fetal T21, T18 or T13 in a single tube reaction on a droplet dPCR platform, as well as its preliminary clinical performance in testing real clinical samples in a clinical environment. By the best of our knowledge at the time this manuscript being prepared, this is the first report to demonstrate that a full dPCR-NIPT technique, consisting of sample processing, cffDNA fraction enriching, the target cell free DNA (cfDNA) concentration enrichments, and dPCR amplification reagents, can simultaneously detect fetal T21, T18 or T13 in maternal plasma samples in a single reaction. The justifications of replacing the SBT with this dPCR-NIPT assay for the upgraded CS as mentioned above are also discussed.
Discussion
By testing the 283 clinical maternal plasma samples in a real clinical environment, we validated the dPCR-NIPT assay on aspects of testing performances, clinical operationality, and user friendliness. With existing dPCR system available on the market, we overcame the challenges of restricted sample volume available and the limited cffDNA fraction and cfDNA concentration presented in the maternal plasma samples by effectively incorporating cffDNA enrichment, specific cfDNA target amplifications and a multiplexed assay design into the dPCR-NIPT assay. The overall test results demonstrated that the dPCR-NIPT assay with a minimum of 18,000 droplets has a significantly statistical power to distinguish aneuploidies from euploid noises with a 95% confidence and is readily applied to screen pregnancies for fetal aneuploidies in clinical. This assay can be further improved in three areas, using a dPCR system with multiple fluorescent detection channels and minimum of 80,000 PCR partitions, increasing plasma sample volume to 4 mL, and continuously optimizing the testing reagents to increase accuracy and reduce variations. A dPCR system with at least 80,000 PCR partitions can increase the test confidence to 99.9% when estimated by Eq.
6 using the same parameters as described above but with a k = 3. Obviously, increasing sample volume can provide higher cfDNA target concentrations leading to a higher PDR to the assay, and further optimizing the testing reagents can enhance the test performances. In addition, upgrading these three areas will increase the test’s precision, which plays a critical role in the dPCR assay. However, even with the current format described in this study, the dPCR-NIPT assay is still a valuable test for screening fetal aneuploidies.
NGS-NIPT is a very powerful tool for screening fetal aneuploidies with high test accuracy [
27‐
29], the main reason why it has not been widely used as a first-tier screen method is its relatively high price [
32‐
35]. The CS has been proposed and used in some counties for purposes of increasing the positive detection rate and reducing the overall costs involved with the NGS assay. In this strategy, pregnant women are first screened by the SBT with a reduced risk cut-off, and then those with a risk rate ≥ 1/1000 were further tested by the NGS-NIPT for confirmations. Since the NGS-NIPT assay only tests the high and intermediate risk pregnancies funneled by the SBT, the positive detection rate of the existing CS is still fully determined by the less sensitive SBT. With a reduced risk cut-off of 1/1000, the SBT showed a higher positive detection rate with a range of 88.9–94.2% due to more samples were referred to the NGS-NIPT test, but also caused a much higher false positive rate with a range of 11.6–17.8%[
32,
33,
55]. Fortunately, these SBT false positives were eventually balanced by the NGS-NIPT assay, thus the most significant benefit of this strategy is to dramatically reduce the number of invasive CVS/AC procedures crediting to the high specificity of the NGS-NIPT assay. Our data revealed that, even with the lowered risk cut-off of 1/1,000, the SBT still could not detect 1 T21 sample and 1 T18 positive sample, meanwhile, brought 63 additional false positive samples that needed to be rescreened by the NGS-NIPT.
Considering the dPCR-NIPT assay has a better sensitivity and specificity, if using it to replace the SBT in the existing CS, the upgraded version would be more effective with a higher positive detection rate and significantly less false positive samples to be resolved by the NGS-NIPT test. A major concern about to use the NGS-NIPT as the first-tier fetal aneuploidy screening test is lack of cost effectiveness due to its relatively higher price at an average of $570 per test with a range of $200-$1,100, while the SBT price is averaged at $53 with a range of $45-$77, depending on the countries [
31‐
35]. If the price can be reduced to an average of $103 with a range of $61-$200 (depending on countries), the NGS-NIPT becomes cost-effective as the first-tier screen test when compared to the existing CS [
32‐
35]. We estimate that the costs of the dPCR-NIPT assay is less than $100 and can even be close to the SBT price after the dPCR consumables being in a mass production. From the cost effectiveness and general performance points of view, the dPCR-NIPT assay provides an immediate solution for NIPT with a higher positive detection rate towards to the NGS-NIPT and a lower price close to the SBT. We would propose an upgraded version of CS for clinical application: all pregnant women are first screened by the dPCR-NIPT assay for trisomic (T21, T18 and T13) fetuses, the high risk ones are then rescreened by the NGS-NIPT test for confirmations, and finally the NGS confirmed high risk ones are diagnosed by the CVS/AC tests. To evaluate the cost effectiveness, we created a simulation situation to estimate and compare the costs for the three screening strategies (i.e., existing CS, the NGS-NIPT as first-tier screen strategy and the upgraded CS) with the following assumptions: 1) 10,000 samples to be tested by each strategy, 2) the average price, as mentioned above, for each of the three first-tier screen methods to be used, and 3) the average false positive rate of 15% for the SBT[
32,
33,
55] and 4.88% for dPCR-NIPT to be used. In this simulation exercise, the upgraded CS had the lowest total costs and could save $107,980 (7.8%) and $4,422,980 (77.6%) when compared to the existing CS and the NGS-NIPT as the first-tier screen strategy, respectively (Table
7). The total cost difference between the existing CS and the upgraded CS seems marginal, however, the latter will provide substantial clinical benefits by enormously increasing the positive detection rate at a lower total cost. It should be mentioned that the turnaround time (TAT) for the existing CS and upgraded CS are comparable, since the NGS-NIPT test used in both CS for the high risk confirmation has a much longer TAT (above 20 h) and makes the time difference between the two primary screen methods negligible. From both the test performance and the cost effectiveness points of view, the dPCR-NIPT assay would be a better solution as a primary screen technology for screening fetal euploidies.
Table 7
Cost comparison of different strategies for screening fetal aneuploidies
Existing CS | 10,000 | STB | 53 | 530,000 | 1500 | NGS-NIPT | 855,000 | 1,385,000 |
NGS-NIPT | 10,000 | NGS-NIPT | 570 | 5,700,000 | – | – | – | 5,700,000 |
Upgraded CS | 10,000 | dPCR-NIPT | 100 | 1,000,000 | 486 | NGS-NIPT | 277,020 | 1,277,020 |
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