Variability in Fetal Fraction Estimation: Comparing Fetal Fractions Reported by Noninvasive Prenatal Testing Providers Globally.

BACKGROUND: Fetal fraction (FF) measurement is considered important for reliable noninvasive prenatal testing (NIPT). Using minimal FF threshold as a quality parameter is under debate. We evaluated the variability in reported FFs of individual samples between providers and laboratories and within a single laboratory. METHODS: Genomic quality assessment and European Molecular Genetics Quality Network provide joint proficiency testing for NIPT. We compared reported FFs across all laboratories and stratified according to test methodologies. A single sample was sequenced repeatedly and FF estimated by 2 bioinformatics methods: Veriseq2 and SeqFF. Finally, we compared FFs by Veriseq and SeqFF in 87 351 NIPT samples. RESULTS: For each proficiency test sample we observed a large variability in reported FF, SDs and CVs ranging from 1.7 to 3.6 and 17.0 to 35.8, respectively. FF measurements reported by single nucleotide polymorphism-based methods had smaller SDs (0.5 to 2.4) compared to whole genome sequencing-based methods (1.8 to 2.9). In the internal quality assessment, SDs were similar between SeqFF (SD 1.0) and Veriseq v2 (SD 0.9), but mean FF by Veriseq v2 was higher compared to SeqFF (9.0 vs 6.4, P 0.001). In patient samples, reported FFs were on average 1.12-points higher in Veriseq than in SeqFF (P 0.001). CONCLUSIONS: Current methods do not allow for a reliable and consistent FF estimation. Our data show estimated FF should be regarded as a laboratory-specific range, rather than a precise number. Applying strict universal minimum thresholds might result in unnecessary test failures and should be used with caution.

Twelve years of assessing the quality of preimplantation genetic testing for monogenic disorders.

OBJECTIVE: Genomics Quality Assessment has provided external quality assessments (EQAs) for preimplantation genetic testing (PGT) for 12 years for eight monogenic diseases to identify sub-optimal PGT strategies, testing and reporting of results, which can be shared with the genomics community to aid optimised standards of PGT services for couples. METHOD: The EQAs were provided in two stages to mimic end-to-end protocols. Stage 1 involved DNA feasibility testing of a couple undergoing PGT and affected proband. Participants were required to report genotyping results and outline their embryo testing strategy. Lymphoblasts were distributed for mock embryo testing for stage 2. Submitted clinical reports and haplotyping results were assessed against peer-ratified criteria. Performance was monitored to identify poor performance. RESULTS: The most common testing methodology was short tandem repeat linkage analysis (59%); however, the adoption of single nucleotide polymorphism-based platforms was observed and a move from blastomere to trophectoderm testing. There was a variation in testing strategies, assigning marker informativity and understanding test limitations, some clinically unsafe. Critical errors were reported for genotyping and interpretation. CONCLUSION: EQA provides an overview of the standard of preimplantation genetic testing-M clinical testing and identifies areas of improvement for accurate detection of high-risk embryos.

One byte at a time: evidencing the quality of clinical service next-generation sequencing for germline and somatic variants.

Next-generation sequencing (NGS) is replacing other molecular techniques to become the de facto gene diagnostics approach, transforming the speed of diagnosis for patients and expanding opportunities for precision medicine. Consequently, for accredited laboratories as well as those seeking accreditation, both objective measures of quality and external review of laboratory processes are required. External quality assessment (EQA), or Proficiency Testing (PT), can assess a laboratory's service through an independent external agency, the EQA provider. The analysis of a growing number of genes and whole exome and genomes is now routine; therefore, an EQA must be delivered to enable all testing laboratories to participate. In this paper, we describe the development of a unique platform and gene target independent EQA scheme for NGS, designed to scale from current to future requirements of clinical diagnostic laboratories testing for germline and somatic variants. The EQA results from three annual rounds indicate that clinical diagnostic laboratories are providing an increasingly high-quality NGS service and variant calling abilities are improving. From an EQA provider perspective, challenges remain regarding delivery and performance criteria, as well as in analysing similar NGS approaches between cohorts with meaningful metrics, sample sourcing and data formats.

Ensuring high standards for the delivery of NIPT world-wide: Development of an international external quality assessment scheme.

OBJECTIVE: To ensure accurate and appropriate reporting of non-invasive prenatal testing (NIPT) results, the standard of testing should be measured and monitored by participation in external quality assessment (EQA) schemes. The findings from international pilot EQAs for NIPT for the common trisomies are presented. METHODS: In the first pilot, three EQA providers used artificially manufactured reference materials to deliver an EQA for NIPT. The second pilot used clinically collected maternal plasma samples. The testing and reporting for aneuploidy status was performed by participating laboratories using routine procedures. Reports were assessed against peer ratified criteria and EQA scores were returned to participants. RESULTS: Forty laboratories participated in the first. Genotyping accuracy was high; four laboratories reported a critical genotyping error (10%) and two reported partial results. Eighty seven laboratories participated in the second pilot using maternal plasma, two reporting a critical genotyping error (2.3%). For both rounds, report content was variable with key information frequently omitted or difficult to identify within the report. CONCLUSIONS: We have successfully delivered an international pilot EQA for NIPT. When compared with currently available manufactured materials, EQA for NIPT was best performed using clinically collected maternal plasma. Work is required to define and improve the standard of reporting.

Recommended practice for laboratory reporting of non-invasive prenatal testing of trisomies 13, 18 and 21: a consensus opinion.

OBJECTIVE: Non-invasive prenatal testing (NIPT) for trisomies 13, 18 and 21 is used worldwide. Laboratory reports should provide clear, concise results with test limitations indicated, yet no national or local guidelines are currently available. Here, we aim to present minimum best practice guidelines. METHODS: All laboratories registered in the three European quality assurance schemes for molecular and cytogenetics were invited to complete an online survey focused on services provided for NIPT and non-invasive prenatal diagnosis. Laboratories delivering NIPT for aneuploidy were asked to submit two example reports; one high and one low risk result. Reports were reviewed for content and discussed at a meeting of laboratory providers and clinicians held at the ISPD 2016 conference in Berlin. RESULTS: Of the 122 laboratories that responded, 50 issued reports for NIPT and 43 of these submitted sample reports. Responses and reports were discussed by 72 attendees at the meeting. Consensus opinion was determined in several areas and used to develop best practice guidelines for reporting of NIPT results. CONCLUSIONS: Across Europe, there is considerable variation in reporting NIPT results. Here, we describe minimum best practice guidelines, which will be distributed to European laboratories, and reports audited in subsequent external quality assurance cycles. © 2017 The Authors. Prenatal Diagnosis published by John Wiley & Sons, Ltd.