Reasons why it comes out as undecidable

Less likely to be ‘uninspectable’

The chances of being ‘untestable’ in our clinics are very low.

Failure to test due to low fetal-derived cell-free (cfDNA) ratio (Fetal Fraction: FF) is usually said to occur 1-8% of the time, and tests are successful in more than 85% of cases retested at our clinics.※1

The blood sample is re-collected after a thorough review of the technical problems in the test and further scrutinised to rule out any possible origin in the quality of the specimen. At the same time, the number of FFs is expected to increase due to the advancing weeks of pregnancy.

Therefore, most cases of inability to test in our clinics are due to biological reasons originating from the specimen and are very rare.

‘Uninspectable’ because of low FF

The majority of the reasons for the inability to test so far have been due to the FF being below the reference value in retests as well.

It is also estimated that 50-60% of women who fail to get a test result the first time due to low FF can be successfully tested by re-collecting blood and retesting.※2,3
For reference, our data show that the test is successful in more than 85% of cases when blood is re-collected after about four weeks.

When does low FF occur?

How is FF calculated in the first place
? FF = foetal cfDNA / (foetal cfDNA + maternal cfDNA )
. As can be seen from this formula, it is a function involving both the amount of maternal cfDNA and foetal cfDNA.

The average FF between 10 and 20 weeks’ gestation is said to be 10-15%. ※4
This means that for whatever reason

• Less foetal cfDNA in maternal blood
• High maternal cfDNA in maternal blood

Reasons for low FF and inspection flow when it occurs

The reasons for each of these are manifold, but the main reasons known so far are summarised below.
(This is limited to those for which academic evidence has been stated at the time of writing the article.)
This is also the actual flow that laboratories follow when low FF levels are detected.

Eliminates the possibility of technical problems

First, it eliminates the possibility of technical problems occurring during the inspection.
This can be established numerically by ensuring that there are no problems with the overall sequence data of interest (typically 45 to 95 sequences are performed simultaneously in a single test) and by re-analysing more detailed bioinformatics data for individual specimens (even verifying quality control items that are not necessary if only to produce results). The results can be verified numerically. Surprisingly few technical problems are eliminated at the inspection stage, as many nowadays follow an automated inspection process and any technical problems are immediately revealed as concrete numerical data in the quality control process.

Elimination of possibilities due to specimen quality

The next step is to eliminate possible causes of specimen origin, such as transport or storage, which may have resulted in specimens that were not testworthy.
This is one of the main reasons why we ask you to re-collect blood. By re-collecting the blood, the fetal cfDNA will be increased, as will be explained later, but at the same time, we will re-evaluate the possibility that there were some irregularities in the quality of the specimen during the first test, such as transport or storage.

In many cases, the laboratory can guarantee to rule out the above by a process of elimination, but with regard to “biological reasons originating from the specimen”, which will be discussed further on, it is only possible to suggest the possibility and it is practically impossible to pinpoint the cause.
(and often not a very meaningful pursuit).

Biological reasons for specimen origin

• Insufficient weeks of gestation (FF increases with advancing weeks)※5
• Lower values of CRL (increasing FF with increasing head length)※6
• Mosaic phenomenon (FF decrease)※7
• Fetus has aneuploidy (effects on FF vary)※8,9,10,11
• Triploid (reduced FF)※12
• Multiple pregnancies (increased total FF, but decreased FF when viewed per foetus)※13,14

(Hereafter, biological reasons of maternal origin)

• Higher maternal BMI values (the higher the higher the FF decrease)※8,5,15
• Maternal autoimmune disease (the more active the condition, the more FF decreases)※16,17,18
• Low molecular weight heparin medication (may decrease FF)※19,20,21,22
• Pregnancy-associated plasma protein PaPP-A in serum (increased FF)※6,23
• Free β-HCG in serum (increased FF)※6,24
• Ethnic differences (impact on FF varies)※6,14,25
• Conception through assisted reproductive technology ART (FF reduction)※24
• Number of transits (FF decrease)※14, 25
• Maternal age (FF decrease)※14, 25

Limiting this biological reason is unfortunately almost impossible, as mentioned above.
It would take a great deal of time and money to carry out a thorough and professional examination of each possibility, which would be impractical, and even if a cause could be identified, all that would be known is that in many cases the NIPT test would not be suitable for the pregnant woman in question.

【References】

• Yaron Y. The implications of non-invasive prenatal testing failures: a review of an under-discussed phenomenon. Prenat Diagn. 2016;36:391-396.
• Kinnings SL, Geis JA, Almasri E, et al. Factors affecting levels of circulating cell-free fetal DNA in maternal plasma and their implications for noninvasive prenatal testing. Prenat Diagn. 2015;35:816-822.
• Hui L, Teoh M, da Silva Costa F, et al. Clinical implementation of cell free DNA based aneuploidy screening: perspectives from a national audit. Ultrasound Obstet Gynecol. 2015;45:10-15.
• Ashoor G, Syngelaki A, Poon LC, Rezende JC, Nicolaides KH. Fetal fraction in maternal plasma cell-free DNA at 11-13 weeks’ gestation: relation to maternal and fetal characteristics. Ultrasound Obstet Gynecol. 2013;41:26-32.
• Kinnings SL, Geis JA, Almasri E, et al. Factors affecting levels of circulating cell-free fetal DNA in maternal plasma and their implications for noninvasive prenatal testing. Prenat Diagn. 2015;35:816-822.
• Ashoor G, Syngelaki A, Poon LC, Rezende JC, Nicolaides KH. Fetal fraction in maternal plasma cell-free DNA at 11-13 weeks’ gestation: relation to maternal and fetal characteristics. Ultrasound Obstet Gynecol. 2013;41:26-32.
• Brison N, Neofytou M, Dehaspe L, et al. Predicting fetoplacental chromosomal mosaicism during noninvasive prenatal testing. Prenat Diagn. 2018;38(4):258-266. https://doi.org/10.1002/pd.5223.
• Ashoor G, Syngelaki A, Poon LC, Rezende JC, Nicolaides KH. Fetal fraction in maternal plasma cell-free DNA at 11-13 weeks’ gestation: relation to maternal and fetal characteristics. Ultrasound Obstet Gynecol. 2013;41:26-32.
• Kinnings SL, Geis JA, Almasri E, et al. Factors affecting levels of circulating cell-free fetal DNA in maternal plasma and their implications for noninvasive prenatal testing. Prenat Diagn. 2015;35:816-822.
• Palomaki GE, Kloza EM, Lambert-Messerlian GM, et al. Circulating cell free DNA testing: are some test failures informative? Prenat Diagn. 2015;35:289-293.
• Rava RP, Srinivasan A, Sehnert AJ, Bianchi DW. Circulating fetal cell free DNA fractions differ in autosomal aneuploidies and monosomy X. Clin Chem. 2014;60:243-250.
• Nicolaides KH, Syngelaki A, del Mar Gil M, Quezada MS, Zinevich Y. Prenatal detection of fetal triploidy from cell-free DNA testing in maternal blood. Fetal Diagn Ther. 2014;35(3):212-217.
• Hedriana H, Martin K, Saltzman D, Billings P, Demko Z, Benn P. Cell Free DNA fetal fraction in twin gestations in single nucleotide polymorphism-based non-invasive prenatal screening. Prenat Diagn. 2019. https://doi.org/10.1002/pd.5609 [Epub ahead of print].
• Galeva S, Gil MM, Konstantinidou L, Akolekar R, Nicolaides KH. First trimester screening for trisomies by cfDNA testing of maternal blood in singleton and twin pregnancies: factors affecting test failure. Ultrasound Obstet Gynecol. 2019;53:804-809.
• Kinnings SL, Geis JA, Almasri E, et al. Factors affecting levels of circulating cell-free fetal DNA in maternal plasma and their implications for noninvasive prenatal testing. Prenat Diagn. 2015;35:816-822.
• Chan RWY, Jiang P, Peng Z, et al. Plasma DNA aberrations in systemic lupus erythematosus revealed by genomic and methylomic sequencing. Proc Natl Acad Sci U S A. 2015;111:E5302-E5311.
• Hui L, Bethune M, Weeks A, Kelley J, Hayes L. Repeated failed non invasive prenatal testing owing to low cell-free fetal DNA fraction and increased variance in a woman with severe autoimmune disease. Ultrasound Obstet Gynecol. 2014;44:242-243.
• Hui CY, Tan WC, Tan EL, Tan LK. Repeated failed non-invasive prenatal testing in a woman with immune thrombocytopenia and antiphospholipid syndrome: lessons learnt. BMJ Case Rep. 2016;2016. https://doi.org/10.1136/bcr-2016-216593.
• Burns W, Koelper N, Barberio A, et al. The association between anti coagulation therapy, maternal characteristics, and a failed cfDNA test due to a low fetal fraction. Prenat Diagn. 2017;37:1125-1129.
• Ma G, Wu W, Lee M, Lin Y, Chen M. Low-molecular-weight heparin associated with reduced fetal fraction and subsequent false-negative cell-free DNA test result for trisomy 21.Ultrasound Obstet Gynecol. 2018;51:276-277.
• Gromminger S, Erkan S, Schock U, et al. The influence of low molecular weight heparin medication on plasma DNA in pregnant women.Prenat Diagn. 2015;35:1155-1157.
• Dabi Y, Guterman S, Jani JC, et al. Autoimmune disorders but not heparin are associated with cell-free fetal DNA test failure. J Transl Med. 2018;16(1):335. https://doi.org/10.1186/s12967-018-1705-2.
• Scott FP, Menezes M, Palma-Dias R, et al. Factors affecting cell-free DNA fetal fraction and the consequences for test accuracy. J Matern Fetal Neonatal Med. 2018;31(14):1865-1872.
• Lee TJ, Rolnik DL, Menezes MA, McLennan AC, da Silva Costa F. Cell-free fetal DNA testing in singleton IVF conceptions. Hum Reprod. 2018;33(4):572-578.
• Rolnik DL, Da Silva Costa F, Lee TJ, Schmid M, McLennan AC. Association between fetal fraction on cell-free DNA testing and first trimester markers for pre-eclampsia. Ultrasound Obstet Gynecol. 2019. https://doi.org/10.1002/uog.18993.