Prenatal screening for aneuploidy has changed dramatically since the 1970s. Non-invasive methods developed in the 1980s and 1990s, combined measurements of maternal serum analytes and ultrasonography. The problem with those methods was not just a high false-negative rate of 12% to 23%, a high positive rate of 5% and a poor sensitivity, ranging from 50% to 95% 1. Uncertain results frequently led to invasive procedures such as amniocentesis or chorionic villi sampling to perform karyotyping on fetal samples. Both of those procedures carry a risk of miscarriage.
The discovery of fetal cells in 19792 and fetal DNA in maternal blood in 19973 were key to the development of an alternative screening method using cell-free fetal DNA (cfDNA) for non-invasive prenatal testing (NIPT). However, it took until 2008 to demonstrate that trisomy can be identified in maternal blood by using next-generation sequencing (NGS)4,5. This opened-up a race to develop the best NIPT tests using NGS to offer as a more accurate and safer method aiming to avoid invasive test follow up.
Identification of extra fetal chromosomes in maternal plasma was originally approached by using massive parallel shotgun sequencing (MPSS) methods combined with chromosome counting. As the method amplifies all the chromosomes, it suffers from variation in amplification efficiency across the genome. This results in variability of aneuploidy detection for different chromosomes. Counting of the chromosomes is detecting a difference in the quantity of fetal vs maternal cfDNA. This difference increases with higher levels of fetal fraction in cfDNA.
In order to decrease redundancy in amplification, targeted sequencing methods were developed for NIPT. This reduced the number of analyzed reads, improved efficiency of analysis and increased sensitivity. Combining targeted sequencing with SNP analysis allowed differentiation between maternal and fetal DNA in a sample. A further push for improved performance of NIPT methods is a shift to pair-end whole-genome sequencing, which allows differentiation of maternal and fetal cfDNA based on fragment sizes. In addition to reducing the required depth of sequencing by two thirds, it is claimed to be much less sensitive to fetal fraction to return a valid result.
The NGS-based methods have been used by NIPT labs since 2011 and despite a decrease in NGS costs, NIPT implementation is still costly. However, the affordability of the tests in developed countries has improved, especially with increased coverage of tests by health insurance. However, the market in developing countries is much more price-sensitive and some companies are actively working on and launching cheaper alternatives. A Swedish company, Vanadis, currently part of PerkinElmer uses a rolling circle amplification on its platform6. A Cyprus based start-up, NIPD Genetics Ltd, patented a methylation-based MeDIP qPCR technique for detecting aneuploidies. And Bio-Rad announced that it works on the digital PCR version for NIPT testing7. We will likely see further technology evolution driving the quality of tests up and costs down.
NIPT test landscape
Following the early papers, many companies started working on NIPT tests and rapidly, within two years seven tests were launched. The field became even more crowded in the next few years with additional tests being developed and launched.
- 2011 – Sequenome launches MaterniT21 Plus
- 2011 – BGI launches NIFTY
- 2011 – Berry Genomics launches BambniTest
- 2012 – Verinata Health launches Verifi test
- 2012 – Ariosa Diagnostics launches Harmony test
- 2012 – LifeCodexx launches PrenaTest
- 2012 - Natera launches Panorama test
- 2014 – LabCorp launches InformaSeq
- 2014 – Sequenome launches VisibiliT – for average-risk pregnancies
- 2015 - Quest launches Q-natal Advanced
- 2017 – Illumina launches VeriSeq NIPT solution
- 2018 – PerkinElmer announces CE-IVD mark for its NIPT Vanadis solution
While this list captures the main tests, new tests are being developed continuously and old tests being improved. Additionally, there has been a dramatic increase in a number of labs offering NIPT tests, either licensed from the companies which created them or developed as LDTs.
Adoption of NIPT in clinical practice
NIPT testing was initially aimed at women at increased risk of aneuploid pregnancies. These were women over the age of 35 or those who previously had a trisomy baby. The official ACOG recommendations in 2013 were that NIPT should be offered only to women within a high-risk group because insufficient evaluations were done in low-risk group8. Within four years, in 2016, ACOG issued new recommendations stating that NIPT should now be offered to all pregnant women. In 2016, ACOG recommends:
“Informing all pregnant women that NIPS is the most sensitive screening option for traditionally screened aneuploidies (i.e., Patau, Edwards, and Down syndromes)”1.
The reason for the change was clinical utility trials comparing NIPT and conventional screening methods for women in average- and high-risk groups. Data has demonstrated that NIPT is superior to the conventional screening method in low-risk women not just in the high-risk group. The studies focused on the three most common trisomies: chromosome 21 (Down Syndrome), 18 (Edwards Syndrome) and 13 (Patau’s Syndrome). This opened the door for a much wider adoption of NIPT.
What is needed to implement NIPT tests?
Implementation of NIPT tests increases globally. While some tests are still mostly carried out by a few central labs, others are being adopted by service labs and smaller clinics. Independent of the size, the labs have to be certain of their ability to identify trisomies reliably. Some of the ACOG guidelines relating to testing itself include the following 1:
- “Laboratories should provide readily visible and clearly stated detection rate (DR), clinical specificity (SPEC), positive predictive value (PPV), and negative predictive value (NPV) for conditions being screened, in pretest marketing materials, and when reporting laboratory results to assist patients and providers in making decisions and interpreting results.
- Laboratories should not offer to screen for Patau, Edwards, and Down syndromes if they cannot report DR, SPEC, and PPV for these conditions
- All laboratories should include a clearly visible fetal fraction on NIPS reports.
- All laboratories should establish and monitor analytical and clinical validity for the fetal fraction.
- All laboratories should specify the reason for a no-call when reporting NIPS results”
How can your lab comply with these guidelines?
The simplest method is to use appropriate NIPT reference materials, which will allow you to report DR, SPEC, PPV, and NPV for each test. Such reference materials will also help you establish that each reported result is within the range established in your lab and its fetal fraction is above the detection cut-off.
At SeraCare we can help you with developing and/or validating your NIPT assay, establishing performance criteria and provide you with tools and materials for monitoring your assay performance. Check out our current offering of NIPT reference materials, especially the new SNP-matched materials compatible with a wide selection of tests.
- Gregg, A. R. et al. Noninvasive prenatal screening for fetal aneuploidy, 2016 update: a position statement of the American College of Medical Genetics and Genomics. Genet. Med. 18, 1056–1065 (2016), PMID: 27467454
- Herzenberg, L. A., Bianchi, D. W., Schröder, J., Cann, H. M. & Iverson, G. M. Fetal cells in the blood of pregnant women: Detection and enrichment by fluorescence-activated cell sorting. Proc. Natl. Acad. Sci. U. S. A. 76, 1453–1455 (1979), PMID: 286330
- Dennis Lo, Y. M. et al. Presence of fetal DNA in maternal plasma and serum. Lancet 350, 485–487 (1997), PMID: 9274585
- Chiu, R. W. K. 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. 105, 20458–20463 (2008), PMID: 19073917
- Fan, H. C., Blumenfeld, Y. J., Chitkara, U., Hudgins, L. & Quake, S. R. Noninvasive diagnosis of fetal aneuploidy by shotgun sequencing DNA from maternal blood. Proc. Natl. Acad. Sci. 105, 16266–16271 (2008), PMID: 18838674
- Dahl, F. et al. Imaging single DNA molecules for high precision NIPT. Sci. Rep. 8, (2018), PMID: 29540801
- Madeleine Johnson. Bio-Rad Expects Expansion of Clinical ddPCR System Menu With FDA Clearance. GenomeWeb (2019). Available at https://www.genomeweb.com/. Accessed 30th January 2020
- ACOG approves new trisomy screen for high-risk pregnancies. Am. J. Med. Genet. Part A 161, vii–ix (2013), PMID: 23436391