A New Safe Blood Test to Diagnose Down Syndrome

14 03 2011

The established method to prenatally diagnose chromosomal gross abnormalities is to obtain fetal cells from the womb with a fine needle, either by Amniocentesis (a sample of the fluid surrounding the foetus in the womb)  or by Chorionic villus sampling (CVS, a sample of the placenta taken via the vaginal route).
The procedures are not to be sneezed at. I’ve undergone both, so I talk from experience. It is kind of horrifying to see a needle entering the womb near to your baby, also because you realize that there is a (small) chance that the procedure will cause a miscarriage. Furthermore, in my case  (rhesus negative) I also had to get an injection of human anti-D immunoglobulin as a precaution to prevent rhesus disease after birth. Finally it takes ages (ok 2-3 weeks) to hear the results. At that point the fetus is already 14-18 weeks old and there is little time to intervene, if that is what is decided.

en: Karyotype of 21 trisomy (Down syndrome) fr...

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Over the years many non-invasive alternatives have been sought to test for Down Syndrome, the most common chromosomal abnormality,  affecting chromosome 21. Instead of one pair of  chromosomes, there is (usually) a third chromosome 21 (hence trisomy 21) (see Fig).

An older non-invasive test is a simple blood test to check the levels of some proteins and hormones in the mother’s blood, that are somehow related to Down Syndrome. However, this test is not very accurate.

The same is true for another non-invasive method: the ultrasound scan of the neck of the fetus. An increased amount of translucent fluid behind the neck (‘nuchal translucency‘) is associated with Down syndrome and a few other chromosomal defects.

A combination of serum tests and nuchal translucency in the 11th week correctly identifies fetuses with Down syndrome 87 percent of the time, whereas it misidentifies healthy fetuses as having Down Syndrome in 5% of the cases (5% false positive rate).

For this reason these non-invasive tests are usually used to “screen”, not to diagnose trisomy 21.

Ever since circulating fetal cells and free fetal DNA were found in the maternal blood, researchers try to enrich for this fetal sources and try to characterize chromosomal aberrations, using very sensitive molecular diagnostic tools, like polymerase chain reaction PCR (i.e. see this post) . The first attempts were directed at detecting the Y chromosome of male babies in the blood of the mother [1].

This January, Chiu et al published an article in the BMJ showing that Down’s syndrome can be detected with greater than 98% accuracy in maternal blood [2]. The group of Lo tested 753 pregnant women at high risk for carrying a fetus with trisomy 21 with this new blood test and compared it with the results obtained by karyotyping (analyzing number and appearance of chromosomes). The new technique is called multiplexed massively parallel DNA sequencing. This is a high throughput sequencing technique in which many DNA-fragments are sequenced in parallel. Sequencing means that the genetic code is unraveled. It is even possible to analyze 2 to 8 labeled maternal samples in parallel (2- and 8-plex reaction).

This parallel DNA sequencing method is “just” a counting method, in which the overall number of chromosomes is counted and one looks at an overrepresentation of chromosome 21.

With the superior 2-plex approach, 100% of the 86 known trisomy 21 fetuses were detected at a 2.1% false positive rate. In other words  the duplex approach had 100% sensitivity (all known positives were detected) and 97.9% specificity (2 were positive according to the test, whereas they were not in reality).

Thus it is a good and non-invasive technique to exclude Down syndrome in pregnant women known to have a high risk of Down syndrome. The approach might perform less well in a low risk group. Furthermore the study was not fully blinded. A practical disadvantage of this new test is that it is expensive, requiring machines not yet available in most hospitals (A spoonful of Medicine).

Another approach, recently published in Nature Medicine doesn’t have this disadvantage [3]. It involves the application of methylated DNA immunoprecipitation (MeDiP) and real-time quantitative PCR (rt-qPCR), which are accessible in all basic diagnostic laboratories. MedDiP is a technique to enrich for methylated DNA sequences, which are more preponderant in the fetus. Next rt-qPCR (amplification of DNA) is used to assess whether the fetus has an extra copy of the fetal-specific methylated region compared to a normal fetus.

In an initial series of 20 known normal cases and 20 known trisomy 21 cases, the researchers tested several differentially methylated regions (DMRs).  The majority of the ratio values in normal cases were at or below a value of 1, whereas in trisomy 21 cases the ratio values were above a value of 1. A combination of 8 specific DMRs out of 12 enabled the correct diagnosis of all the cases.

Next the authors validated the technique by applying the above method to 40 new samples in a blinded fashion. These samples contained 26 normal cases and 14 trisomy 21 cases (as later defined by karyotyping). Normal and trisomy 21 cases were all correctly identified.

The authors conclude that they achieved 100% sensitivity and 100% specificity in 80 samples. However, the first 40 samples were used to calibrate the test, thus the real validation study was done in a small set of 40, containing only 14 trisomy cases. One can imagine that a greater sample could have a few more false negatives or false positives. Indeed, small initial studies are likely to overestimate the true effect.

Furthermore, there was an overrepresentation of trisomy 21 cases (1/3 of the sample). Thus it is to soon to say that this trisomy 2 method is to be potentially employed in the routine practice of all diagnostic laboratories and be applicable to all pregnancies”, as the authors did. To this end the method should be confirmed in larger studies and in low risk pregnancies.

In conclusion, the relative easy and cheap methylated DNA immunoprecipitation/real-time quantitative PCR combo test, seems a promising approach to screen for Down syndrome in high risk pregnancies. Larger studies are needed to confirm the extreme accuracy of 100% and must demonstrate the applicability to low risk pregnancies. If confirmed, this blood test could eliminate the need for invasive procedures. Another positive aspect is that the test can be performed early, from the 11th week of gestation, and the results can be obtained within 4-5 days. Moreover the researchers can easily adapt the current technique to demonstrate abnormal numbers (aneuploidy) of chromosomes 13, 18, X and Y.


  • LO, Y., CORBETTA, N., CHAMBERLAIN, P., RAI, V., SARGENT, I., REDMAN, C., & WAINSCOAT, J. (1997). Presence of fetal DNA in maternal plasma and serum The Lancet, 350 (9076), 485-487 DOI: 10.1016/S0140-6736(97)02174-0
  • Chiu, R., Akolekar, R., Zheng, Y., Leung, T., Sun, H., Chan, K., Lun, F., Go, A., Lau, E., To, W., Leung, W., Tang, R., Au-Yeung, S., Lam, H., Kung, Y., Zhang, X., van Vugt, J., Minekawa, R., Tang, M., Wang, J., Oudejans, C., Lau, T., Nicolaides, K., & Lo, Y. (2011). Non-invasive prenatal assessment of trisomy 21 by multiplexed maternal plasma DNA sequencing: large scale validity study BMJ, 342 (jan11 1) DOI: 10.1136/bmj.c7401
  • Papageorgiou, E., Karagrigoriou, A., Tsaliki, E., Velissariou, V., Carter, N., & Patsalis, P. (2011). Fetal-specific DNA methylation ratio permits noninvasive prenatal diagnosis of trisomy 21 Nature Medicine DOI: 10.1038/nm.2312

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