Performance qualification

PrenaTest^® for the determination of fetal trisomy 21 (test option 1)

The performance of the PrenaTest^® Option 1 has been validated in a clinical study. The study results of the maternal plasma samples (n=1020) demonstrated a positive percentage agreement (PPA; equates to sensitivity) of 100% (lower 1-sided 95% confidence interval of 93.12%; n=42/42) and a negative percentage agreement (NPA; equates to specificity; lower 1-sided 95% confidence interval of 99.54%; n=1019/1020) of 99.9% compared to NGS-based PrenaTest^®. The negative predictive value (NPV) was 100% (lower 1-sided 95% confidence interval of 99.71%). The PrenaTest^® Option 1 can be applied in the case of singleton pregnancies.

PrenaTest^® Option 2 and 2 Plus as well as Option 3 and 3 Plus

Source: Illumina VeriSeq NIPT Solution v2 Package Insert, Document#1000000078751v01, August 2019

Sensitivity and specificity for detecting trisomies 21, 18 and 13 for singleton pregnancies

	Trisomy 21	Trisomy 18	Trisomy 13
Sensitivity	>99,9% (130/130)	>99,9% (41/41)	>99,9% (26/26)
2-sided 95%-KI	97,1%, 100%	91,4%, 100%	87,1%, 100%
Specificity	99,90% (1.982/1.984)	99,90% (1.995/1.997)	99,90% (2.000/2.002)
2-sided 95%-KI	99,63%, 99,97%	99,64%, 99,97%	99,64%, 99,97%

Estimates for trisomy 21, 18 and 13 in simulated population of twin pregnancies

	Trisomy 21	Trisomy 18	Trisomy 13	Presence of Y
Sensitivity	96,4%	95,7%	93,6%	>99,9%
2-sided 95%-KI	(86,4%, 98,9%)	(68,3%, 99,4%)	(64,1%, 98,9%)	(99,9%, >99,9%)
Specificity	99,9 %	>99,9%	>99,9%	>99,9%
2-sided 95%-KI	(99,8%, >99,9%)	(99,9%, >99,9%)	(99,9%, >99,9%)	(99,7%, >99,9%)

Sensitivity und Specificity for rare autosomal aneuploidy (RAA); including known mosaics

	Sensitivity	Specificity
Estimate % (n/N)	96,4% (27/28)	99,80% (2.001/2.005)
2-sided 95%-KI	82,3%, 99,4%	99,49%, 99,92%

PrenaTest^® for the detection of the 22q11.2 microdeletion (optional for test options 2 oder 3)

Phase 1
A total of 469 samples were tested, of which 175 (37.3%) met the quality criteria. Three positive samples with a 22q11.2 microdeletion were correctly determined (3/3, 100%). All negative samples were correctly classified (172/172, 100%). There were no false-positive or false-negative results.

Phase 2
In a final internal blinded study, 20 samples from Phase 1 were examined. All samples were classified correctly. Due to the low number of cases a concrete test sensitivity and specificity cannot be derived.

Fetal gender determination

For PrenaTest^® Option 1 the determination of the fetal gender is based on the proprietary qPCR assay QuantYfeX^®. The assay applied for fetal gender determination has not been validated in a clinical study; however, it has successfully completed an internal methodological-technical validation, in which 1160 samples were examined. A –male– result is reported, if a Y chromosomal marker (SRY) is detected by QuantYfeX^®. A –female– result is reported, if a Y chromosomal marker (SRY) is not detected by QuantYfeX^®. In very rare cases the fetal gender cannot be determined. In this case the fetal gender is not definable and the analysis will not be repeated.

Literature

• Stumm et al. 2012. Noninvasive prenatal detection of chromosomal aneuploidies using different next generation sequencing strategies and algorithms. Prenatal Diagnosis 2012, 32, 569–577
• Stumm et al. 2013. Diagnostic accuracy of random massively parallel sequencing for non-invasive prenatal detection of common autosomal aneuploidies: a collaborative study in Europe.Prenatal Diagnosis 2013, 33,1 –7
• Groemminger et al. 2014. Fetal Aneuploidy Detection by Cell-Free DNA Sequencing for Multiple Pregnancies and Quality Issues with Vanishing Twins. J. Clin. Med. 2014, 3, 679-692; doi:10.3390/jcm303067
• Floeck et al. 2017. Non-invasive prenatal testing (NIPT): Europe’s first multicenter post-market clinical follow-up study validating the quality in clinical routine. Arch Gynecol Obstet DOI 10.1007/s00404-017-4517-3
  Hofmann et al. 2014. NIPT: Welche Unterschiede zwischen den Tests gibt es tatsächlich? FRAUENARZT 55 (2014) Nr. 11
• Groemminger et al. 2015. The influence of low molecular weight heparin medication on plasma DNA in pregnant women. Prenatal Diagnosis 2015, 35, 1–3
• Wolf et al. 2016. Purification of Circulating Cell-Free DNA from Plasma and Urine Using the Automated Large-Volume Extraction on the QIAsymphony^® SP Instrument. © Springer International Publishing Switzerland 2016 P.B. Gahan et al. (eds.), Circulating Nucleic Acids in Serum and Plasma – CNAPS IX, Advances in Experimental Medicine and Biology 924, DOI 10.1007/978-3-319-42044-8_33