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.

3rd Department of Internal Medicine 1st Faculty of Medicine Charles University and General University Hospital Prague U Nemocnice 1 128 08 Prague 1 Czech Republic

Department of Gynaecology Obstetrics and Neonatology 1st Faculty of Medicine Charles University and General University Hospital Prague Apolinarska 18 128 51 Prague Czech Republic

Department of Laboratory Medicine Faculty of Health Care and Social Work University of Trnava in Trnava Universitne Namestie 1 918 43 Trnava Slovak Republic

Department of Microsystem Engineering School of Mechanical Engineering Northwestern Polytechnical University 127 West Youyi Road Xi'an 710072 Shaanxi People's Republic of China

Department of Nephrology 1st Faculty of Medicine Charles University and General University Hospital Prague U Nemocnice 499 2 128 08 Prague 2 Czech Republic

GENvia Karlovo náměstí 7 128 00 Prague 2 Czech Republic

Institute of Biology and Medical Genetics 1st Faculty of Medicine Charles University and General University Hospital Prague Albertov 4 128 00 Prague Czech Republic

Prenatal Diagnosis Center ProfiG2 S R O Vajgarská 1141 Prague Czech Republic

Zobrazit více v PubMed

Bull MJ. Down syndrome. N. Engl. J. Med. 2020;382:2344–2352. doi: 10.1056/NEJMra1706537. PubMed DOI

Akhtar, F. & Bokhari, S. R. A. Down Syndrome. in StatPearls (StatPearls Publishing, 2022). PubMed

Antonarakis SE, et al. Down syndrome. Nat. Rev. Dis. Primer. 2020;6:9. doi: 10.1038/s41572-019-0143-7. PubMed DOI PMC

Asim A, Kumar A, Muthuswamy S, Jain S, Agarwal S. Down syndrome: An insight of the disease. J. Biomed. Sci. 2015;22:41. doi: 10.1186/s12929-015-0138-y. PubMed DOI PMC

sipek@vrozene-vady.cz, content: Mud. _Antonin_Sipek_CSc ; & admin@vrozene-vady.cz, code: M. A. jr [Azrael]; Vrozené vady - Informační portál o vrozených vadách. http://www.vrozene-vady.cz/prezentace/index.php?co=2022.

Wald NJ, Kennard A, Hackshaw A, McGuire A. Antenatal screening for Down’s syndrome. J. Med. Screen. 1997;4:181–246. doi: 10.1177/096914139700400402. PubMed DOI

Dai P, et al. A dPCR-NIPT assay for detections of trisomies 21, 18 and 13 in a single-tube reaction-could it replace serum biochemical tests as a primary maternal plasma screening tool? J. Transl. Med. 2022;20:269. doi: 10.1186/s12967-022-03455-y. PubMed DOI PMC

Springer D, et al. Importance of the integrated test in the Down’s syndrome screening algorithm. J. Med. Screen. 2018;25:114–118. doi: 10.1177/0969141317752533. PubMed DOI

American College of Obstetricians and Gynecologists’ Committee on Practice Bulletins—Obstetrics, Committee on Genetics, & Society for Maternal-Fetal Medicine. Screening for Fetal Chromosomal Abnormalities: ACOG Practice Bulletin, Number 226. Obstet. Gynecol.136, e48–e69 (2020). PubMed

Gray KJ, Wilkins-Haug LE. Have we done our last amniocentesis? Updates on cell-free DNA for Down syndrome screening. Pediatr. Radiol. 2018;48:461–470. doi: 10.1007/s00247-017-3958-y. PubMed DOI PMC

Bianchi DW, et al. DNA sequencing versus standard prenatal aneuploidy screening. N. Engl. J. Med. 2014;370:799–808. doi: 10.1056/NEJMoa1311037. PubMed DOI

Lo YMD, et al. Presence of fetal DNA in maternal plasma and serum. Lancet. 1997;350:3. doi: 10.1016/S0140-6736(97)02174-0. PubMed DOI

Hui L, Bianchi DW. Fetal fraction and noninvasive prenatal testing: What clinicians need to know. Prenat. Diagn. 2020;40:155–163. doi: 10.1002/pd.5620. PubMed DOI PMC

Chiu RWK, et al. Noninvasive prenatal diagnosis of fetal chromosomal aneuploidy by massively parallel genomic sequencing of DNA in maternal plasma. Proc. Natl. Acad. Sci. U. S. A. 2008;105:20458–20463. doi: 10.1073/pnas.0810641105. PubMed DOI PMC

Shekhawat DS, et al. Critical appraisal of droplet digital polymerase chain reaction application for noninvasive prenatal testing. Congenit. Anom. 2022;62:188–197. doi: 10.1111/cga.12481. PubMed DOI

Zhu H, et al. PCR past, present and future. Biotechniques. 2020 doi: 10.2144/btn-2020-0057. PubMed DOI PMC

El Khattabi LA, et al. Could digital PCR be an alternative as a non-invasive prenatal test for trisomy 21: A proof of concept study. PLoS ONE. 2016;11:e0155009. doi: 10.1371/journal.pone.0155009. PubMed DOI PMC

Tan C, et al. A multiplex droplet digital PCR assay for non-invasive prenatal testing of fetal aneuploidies. The Analyst. 2019;144:2239–2247. doi: 10.1039/C8AN02018C. PubMed DOI

Horinek A, et al. Cell-free fetal DNA in maternal plasma during physiological single male pregnancies: Methodology issues and kinetics. Fetal Diagn. Ther. 2008;24:15–21. doi: 10.1159/000132400. PubMed DOI

Karlin-Neumann, G., Bizouarn, F. (eds.) Digital PCR Methods and Protocols. Methods in Molecular Biology, 1768, (Humana Press, Springer Verlag, Heidelberg, 2018) PubMed

Mandrekar JN. Receiver operating characteristic curve in diagnostic test assessment. J. Thorac. Oncol. 2010;5:1315–1316. doi: 10.1097/JTO.0b013e3181ec173d. PubMed DOI

McKanna T, et al. Fetal fraction-based risk algorithm for non-invasive prenatal testing: Screening for trisomies 13 and 18 and triploidy in women with low cell-free fetal DNA. Ultrasound Obstet. Gynecol. 2019;53:73–79. doi: 10.1002/uog.19176. PubMed DOI PMC

Zhang C, He Q, Qiao L, Li H, Wang T. The performance of grey zone in common foetal aneuploidy screening by semiconductor sequencing. J. Obstet. Gynaecol. 2022;42:1782–1787. doi: 10.1080/01443615.2022.2039902. PubMed DOI

Yu SCY, et al. Size-based molecular diagnostics using plasma DNA for noninvasive prenatal testing. Proc. Natl. Acad. Sci. 2014;111:8583–8588. doi: 10.1073/pnas.1406103111. PubMed DOI PMC

Greytak SR, et al. Harmonizing cell-free DNA collection and processing practices through evidence-based guidance. Clin. Cancer Res. Off. J. Am. Assoc. Cancer Res. 2020;26:3104–3109. doi: 10.1158/1078-0432.CCR-19-3015. PubMed DOI PMC

You Y. Design of LNA probes that improve mismatch discrimination. Nucleic Acids Res. 2006;34:e60–e60. doi: 10.1093/nar/gkl175. PubMed DOI PMC

De Spiegelaere W, et al. Touchdown digital polymerase chain reaction for quantification of highly conserved sequences in the HIV-1 genome. Anal. Biochem. 2013;439:201–203. doi: 10.1016/j.ab.2013.04.024. PubMed DOI

SPEED: an integrated, smartphone-operated, handheld digital PCR Device for point-of-care testing