Dataset Information

Rapid non-invasive prenatal screening test for trisomy 21 based on digital droplet PCR.

ABSTRACT: Non-invasive prenatal tests for the detection of fetal aneuploidies are predominantly based on the analysis of cell-free DNA (cfDNA) from the plasma of pregnant women by next-generation sequencing. The development of alternative tests for routine genetic laboratories is therefore desirable. Multiplex digital droplet PCR was used to detect 16 amplicons from chromosome 21 and 16 amplicons from chromosome 18 as the reference. Two fluorescently labeled lock nucleic acid probes were used for the detection of reaction products. The required accuracy was achieved by examining 12 chips from each patient using Stilla technology. The plasma cfDNA of 26 pregnant women with euploid pregnancies and 16 plasma samples from pregnancies with trisomy 21 were analyzed to determine the cutoff value for sample classification. The test was validated in a blind study on 30 plasma samples from pregnant patients with a risk for trisomy 21 ranging from 1:4 to 1:801. The results were in complete agreement with the results of the invasive diagnostic procedure (sensitivity, specificity, PPV, and NPV of 100%). Low cost, and speed of analysis make it a potential screening method for implementation into the clinical workflow to support the combined biochemical and ultrasound results indicating a high risk for trisomy 21.

SUBMITTER: Lassakova S

PROVIDER: S-EPMC10746715 | biostudies-literature | 2023 Dec

REPOSITORIES: biostudies-literature

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Rapid non-invasive prenatal screening test for trisomy 21 based on digital droplet PCR.

Laššáková Soňa S Šenkyřík Pavel P Pazourková Eva E Hořínek Aleš A Calda Pavel P Břešťák Miroslav M Světnicová Kamila K Neužil Pavel P Korabečná Marie M

Scientific reports 20231222 1

Non-invasive prenatal tests for the detection of fetal aneuploidies are predominantly based on the analysis of cell-free DNA (cfDNA) from the plasma of pregnant women by next-generation sequencing. The development of alternative tests for routine genetic laboratories is therefore desirable. Multiplex digital droplet PCR was used to detect 16 amplicons from chromosome 21 and 16 amplicons from chromosome 18 as the reference. Two fluorescently labeled lock nucleic acid probes were used for the dete ...[more]

PMID: 38135736

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Project description:BackgroundThe first- and second-trimester screening for trisomy 21 (T21) are reimbursed for all pregnant women in Belgium. Using a cut-off risk of 1:300 for T21, about 5% of all pregnant women are referred for definitive prenatal diagnosis using an invasive test, at a sensitivity of (only) 72.5%. The sensitivity and specificity of the non-invasive prenatal test (NIPT) are over 99% but come at a cost of €460 (£373) per test. The objective is to estimate the consequences of introducing NIPT for the detection of T21.MethodsA cost-consequences analysis was performed presenting the impact on benefits, harms and costs. Context-specific real-world information was available to set up a model reflecting the current screening situation in Belgium. This model was used to construct the second and first line NIPT screening scenarios applying information from the literature on NIPT's test accuracy.ResultsIntroducing NIPT in the first or second line reduces harm by decreasing the number of procedure-related miscarriages after invasive testing. In contrast with NIPT in the second line, offering NIPT in the first line additionally will miss fewer cases of T21 due to less false-negative test results. The introduction of NIPT in the second line results in cost savings, which is not true for NIPT at the current price in the first line. If NIPT is offered to all pregnant women, the price should be lowered to about €150 to keep the screening cost per T21 diagnosis constant.ConclusionsIn Belgium, the introduction and reimbursement of NIPT as a second line triage test significantly reduces procedure-related miscarriages without increasing the short-term screening costs. Offering and reimbursing NIPT in the first line to all pregnant women is preferred in the long term, as it would, in addition, miss fewer cases of T21. However, taking into account the government's limited resources for universal reimbursement, the price of NIPT should first be lowered substantially before this can be realised.

Project description:Objective: Cell-free fetal DNA (cffDNA) testing has opened new options in prenatal screening for trisomy 21. Due to the higher costs of cffDNA testing there is an ongoing debate on how to combine different screening strategies. Methods: For this study, a model-based approach was used to evaluate all births in Germany in 2012 together with the percentage of euploid and trisomic pregnancies. Detection rates (DR), false positive rates (FPR), the costs of different screening strategies for trisomy 21 and combinations of these strategies were compared. The number of fetuses with trisomy 21 at 12 + 0 weeks of gestation was estimated based on maternal age distribution. We examined the screening performance of a screening strategy based on maternal age, first trimester screening (FTS) and cffDNA testing as well as the combinations "maternal age and cffDNA" and "FTS and cffDNA". Results: In 2012 673 544 children were born. Median maternal age at delivery was 30.2 years (25th-75th quartile: 27.0-34.0). Based on maternal age distribution the expected number of fetuses with trisomy 21 at 12 weeks' gestation was 1788. Our study population therefore consisted of 675 332 pregnancies. Screening based only on maternal age or FTS or cffDNA resulted in detection rates of 63.3 %, 92.2 % and 99.0 % and false positive rates of 21.8 %, 8.0 % and 0.1 %, respectively. When maternal age was combined with cffDNA, cffDNA testing was only offered to women over a certain age; if a cut-off of 30 years was used, this resulted in a DR of 85.2 % and a FPR of 1.7 %. If primary screening consisted of FTS with cffDNA testing only done when the risk was between 1 : 10 and 1 : 1000, the detection rate was 96.7 % and the false positive rate was 1.2 %. Conclusion: In this model-based study we showed that prenatal screening for trisomy 21 can be improved even more by combining FTS and cffDNA. Further studies are necessary to examine whether these results can be reproduced in reality.

Project description:BackgroundScreening tests for Trisomy 21 (T21), also known as Down syndrome, are routinely performed for the majority of pregnant women. However, current tests rely on either evaluating non-specific markers, which lead to false negative and false positive results, or on invasive tests, which while highly accurate, are expensive and carry a risk of fetal loss. We outline a novel, rapid, highly sensitive, and targeted approach to non-invasively detect fetal T21 using maternal plasma DNA.Methods and findingsHighly heterozygous tandem Single Nucleotide Polymorphism (SNP) sequences on chromosome 21 were analyzed using High-Fidelity PCR and Cycling Temperature Capillary Electrophoresis (CTCE). This approach was used to blindly analyze plasma DNA obtained from peripheral blood from 40 high risk pregnant women, in adherence to a Medical College of Wisconsin Institutional Review Board approved protocol. Tandem SNP sequences were informative when the mother was heterozygous and a third paternal haplotype was present, permitting a quantitative comparison between the maternally inherited haplotype and the paternally inherited haplotype to infer fetal chromosomal dosage by calculating a Haplotype Ratio (HR). 27 subjects were assessable; 13 subjects were not informative due to either low DNA yield or were not informative at the tandem SNP sequences examined. All results were confirmed by a procedure (amniocentesis/CVS) or at postnatal follow-up. Twenty subjects were identified as carrying a disomy 21 fetus (with two copies of chromosome 21) and seven subjects were identified as carrying a T21 fetus. The sensitivity and the specificity of the assay was 100% when HR values lying between 3/5 and 5/3 were used as a threshold for normal subjects.ConclusionsIn summary, a targeted approach, based on calculation of Haplotype Ratios from tandem SNP sequences combined with a sensitive and quantitative DNA measurement technology can be used to accurately detect fetal T21 in maternal plasma when sufficient fetal DNA is present in maternal plasma.

Dataset Information

Rapid non-invasive prenatal screening test for trisomy 21 based on digital droplet PCR.

Publications

Rapid non-invasive prenatal screening test for trisomy 21 based on digital droplet PCR.

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