Accuracy and reproducibility of fetal‐fraction measurement using relative quantitation at polymorphic loci with microarray

Abstract

ObjectivesVarious methods of fetal‐fraction measurement have been employed in conjunction with different approaches to cell‐free DNA testing for fetal aneuploidy. In this study, we determined the accuracy and reproducibility of fetal‐fraction measurement using polymorphic assays that are incorporated into the test design as part of the Harmony® prenatal test and evaluated whether the single nucleotide polymorphisms selected for and used in these assays can be applied broadly to all patient populations.MethodsClinical maternal plasma samples were assayed using a custom microarray with Digital ANalysis of Selected Regions (DANSR) assays designed to cover non‐polymorphic targets on chromosomes of interest for aneuploidy assessment (13, 18, 21, X and Y) and polymorphic targets for fetal‐fraction assessment. In a consecutive series of 47 512 maternal plasma samples, fetal‐fraction measurements based on polymorphic assays were compared with those from Y‐sequence quantitation. Reproducibility was examined between first‐ and second‐tube measurements for the same patient sample in 734 cases. The fraction of informative loci was calculated for 13 988 samples.ResultsThere was a strong correlation between fetal fractions determined using the polymorphic assays and using Y‐chromosome sequence quantitation (r = 0.97). Fetal‐fraction measurement between the first and second tubes was highly reproducible (r = 0.98). The fraction of informative loci observed in a clinical series was consistent with predictions based on assay design.ConclusionsThe method based on relative quantitation at polymorphic loci on a microarray is accurate and reproducible for fetal‐fraction estimation and is equally informative across global populations. This study provides a useful benchmark for ensuring the reliability and accuracy of fetal‐fraction measurement. © 2018 Roche Sequencing Solutions. Ultrasound in Obstetrics & Gynecology published by John Wiley & Sons Ltd on behalf of the International Society of Ultrasound in Obstetrics and Gynecology.

Hemolysis and Fetal Fraction in Cell-Free DNA Blood Collection Tubes for Noninvasive Prenatal Testing

“…Specimens were analyzed with the Harmony prenatal test [14,15]. The test, which is intended for determination of the probability of fetal trisomy, uses non-polymorphic digital analysis of selected regions (DANSR) assays on chromosomes 21, 18, and 13 and polymorphic DANSR assays for fetal fraction determination [16].…”

“…In contrast, since fetal cfDNA makes up only a minor fraction of the cfDNA present in maternal plasma, detection of subtle changes in fetal fraction is important. Rather than quantitative polymerase chain reaction, which has been used in previous studies [11], we used single nucleotide polymorphism-based fetal fraction determination, which has been shown to be highly accurate and reproducible [16]. It should be noted that the conclusion that hemolysis is not associated with lower fetal fraction and should not be a reason for specimen rejection was clearly demonstrated in the context of Roche Cell-Free DNA Collection Tubes and the Harmony prenatal test.…”

Fetal fraction and noninvasive prenatal testing: What clinicians need to know

“…However, the methods used to measure FF vary considerably and are not directly comparable, leading to calls for industry standardization in methods and reporting . While Y chromosome‐based methods appear to be the closest to an accepted “gold standard" in this field, these are obviously limited by their applicability to only those pregnancies with a male fetus. Some advocate using a combination of methods to check for the presence of fetal cfDNA and FF calculation for female and male bearing pregnancies .…”

Update on noninvasive prenatal testing: A review based on current worldwide research

“…There are two main methods of NIPT: the aforementioned MPS method and the selective amplification of only a specific region of the target chromosome and then performing NGS. Furthermore, regarding the detection of autosomal trisomies (involving chromosomes 21, 18 and 13), low-cost methods are being developed, such as selectively amplifying only a specific region of the target chromosome and analyzing it using microarrays, 49 quantitative PCR analysis and selectively coloring and quantifying only a specific region of the target chromosome. 50 Furthermore, tests for detecting monogenic diseases have also been clinically applied, and the number of target diseases is increasing.…”