NIPT-Non Invasive Prenatal Testing by utilizing massively parallel sequencing (MPS)

 

What is meant   by digital polymerase chain reactions (PCR) and massively parallel sequencing (MPS)????

What do we mean by cell free foetal DNA –used for detection of  A) aneuploidy of foetus in utero B)  sex determination of  foetus C) and Rh status of foetus. A scientific breakthrough came in 1997 with the recognition that it was not necessary to detect intact fetal cells but(that maternal plasma contained within it fetal cell-free DNA (cfDNA) . It has been shown that this material mainly originates from fetal trophoblast and consists of relatively short fragments of fetal DNA (143 base pairs on average). Recent work has demonstrated that cfDNA represents the whole of the fetal genome than intact fetal cells.

Firstly, cfDNA of fetal origin is present in relatively large quantities, representing up to 20% of the total maternal plasma DNA in later pregnancy.

 

It is also found from early in pregnancy, even from 4-5 weeks’ gestation.

Furthermore, it is very rapidly cleared from the maternal circulation (with a half-life of 16 minutes) making it no longer detectable only hours after delivery. The challenge is that fetal DNA still only represents a minority partner, mixed amongst the maternal DNA, and as such deciphering the fetal from the maternal signature is the major technical challenge.

 

However, technological advances such as digital polymerase chain reactions (PCR) and massively parallel sequencing (MPS), which enable the far more precise enumeration of genes present in cfDNA, now mean  that the far more difficult challenge of identifying a fetus with aneuploidy or even the sequencing of the entire fetal genome are now becoming a reality.

One of the best established, and now widely available/prenatal diagnostic tests on cfDNA,

 enable appropriate counseling and an informed decision regarding whether a definitive diagnostic test is required.  This test may also be important in a range of other clinical
 review and meta-analysis of the diagnostic accuracy of this test which included 90 studies (9965 pregnancies) demonstrated that in the first trimester the test is 95.0% sensitive and 98.8% specific, with this increasing to 98.2% sensitivity and 99.5% specificity in the 2nd trimester . The reason for this increasing accuracy at later gestations is the increase in the level of cfDNA in the maternal circulation . Before  7 weeks the quantum of foetal DNA is too low ., so  the test is not as reliable due to the lower levels of fetal DNA increasing the risk of false negative result . 

The advent of mentors to ensure adequate quantities of fetal DNA are precept in the te sample before testing is undertaken should reduce this false negative rate.(fn addition, accurate dating of the pregnancy is required to ensure the test is not undertaken too early, and ultrasound exclusion of a twin pregnancy or a ‘vanishing twin’ is also important as this The demonstration of the accuracy of prenatal diagnosis of fetal RhD status from cfDNA from large studies has led to their widespread use in clinical practice. In pregnancies at high risk of rhesus disease, such as women already sensitised, knowledge of the fetal RhD status is crucial for management. For these women in settings where these tests are available they have virtually replaced invasive diagnostic procedures to determine fetal RhD status . The area where uncertainty remains is regarding the implementation of such a testing strategy in a law-risk population, the practicalities of integrating such an effort into individual national health care and screening systems, and the cost benefit of such approaches. Indeed, routine testing at 25 weeks’ gestation has already been successfully introduced into antenatal care in Denmark. In the UK recent guidance from the National Institute of Health and Clinical Excellence has recommenced the exploration of routine antenatal fetal RhD typing