Cell free fetal DNA


Refers to fetal DNA circulating freely in the maternal blood stream.

Analysis of cffDNA by sampling mother’s blood provides a method of non-invasive prenatal diagnosis and testing.

Cell free fetal DNA shedding into maternal bloodstream originates from the trophoblasts making up the placenta.

Circulating fetal DNA, cfDNA, is derived primarily from the fragmentation and release of genomics DNA from placental cells.

Cell free-cf DNA is also released during the routine turnover of a persons normal cells.

Assay of the fetal cfDNA is the basis of non-invasive prenatal testing for fetal chromosomal, aneuploidy and monogenic diseases.

Cel free-DNA’s other sources include tumors, transplanted organs, and infectious organisms.

Cell free DNA is useful for the diagnosis in monitoring of cancer, the assessment of transplant-graft rejection, and the diagnosis of infectious diseases.

It is estimated that 2-6% of the DNA in the maternal blood is of fetal origin.

The fetal DNA is fragmented and makes its way into the maternal bloodstream by shedding of the placental microparticles into the maternal bloodstream.

cffDNA can first be observed as early as 7 weeks gestation.

Amount of cfDNA increases as the pregnancy progresses.

cffDNA diminishes quickly after the birth of the baby, so that it is no longer detectable in the maternal blood approximately 2 hours after birth.

cffDNA is significantly smaller than the maternal DNA in the bloodstream, with fragments approximately 200bp in size.

cffDNA screening relies on the presence of small fragments of fetal DNA, typically smaller than 150 base pairs, that freely cross the placenta into the maternal bloodstream beginning early in the first trimester.

cfDNA can be identified as being a fetal n origin by analyzing patterns of single nucleotide polymorphisms, that are unique for the fetus’ genome and can be distinguished from the unique single nucleotide polymorphisms pattern characteristic of maternal DNA that, in turn is extracted from maternal leukocytes.

These tests can be done earlier than the current prenatal testing methods, and have no risk of spontaneous abortion, unlike current prenatal testing methods.

Cell free fetal DNA screening has been approved to determine fetal aneuploidy and screen for fetal aneuploidy, including trisomies 13, 18, and 21, in high-risk and the average risk pregnancies.

It has a high specificity of more than 99.9% for all three trisomies

Non-invasive prenatal diagnosis (NIPD) and Non-invasive prenatal testing (NIPT) have clear benefits above the standard tests of chorionic villi sample (CVS), amniocentesis,and other diseases which have procedure-related miscarriage risks of about 1 in 100 pregnancies and 1 in 200 pregnancies, respectively.

Cell-free fetal DNA techniques share the same ethical and practical issues, such as the possibility of prenatal sex discernment and sex selection.

A sample of the mother’s blood is taken after 10 weeks of pregnancy, although fetal blood can be detected as early as the fifth week.

The test measures the relative amount of free fetal DNA in the mother’s blood which consists of approximately 2-6% of the total:it accounts for approximately 10% of all Crf DNA in maternal serum for the second trimester.

Limitations of the study include the concentration of all cell-free DNA in maternal blood is low, the total amount of cell-free DNA varies between individuals, cell free fetal DNA molecules are out-numbered by maternal cell-free DNA molecules, the fetus inherits half the genome from the mother.

Addition of formaldehyde to maternal blood samples increases the percentage of free fetal DNA by stabilizing intact cells, and inhibit further release of maternal DNA.

The mean percentage of free fetal DNA in maternal bood ranges from 0.32% to 40%, with a mean percentage of 7.7 without formaldehyde-treatment.

The mean percentage of free fetal DNA with formaldehyde treatment is 20.2%.

Fetal DNA is smaller in size, a standardized size fractionation can comprise up to 70% of total cell-free DNA.

To detect fetal DNA, the majority of studies focus on detecting paternally inherited sequences.

Real-time PCR assays for single cell analysis have been developed for a Y-chromosome marker; a common Tay-Sachs disease mutation, the most common cystic fibrosis mutation, and a wide range of thalassemia mutations and Hb S.

Point mutations, copy number variations, loss of heterozygosity or aneuploidy can be detected, and digital PCR can differentiate between maternal blood plasma and fetal DNA in a multiplex fashion.

Able to identify aneuploid pregnancies, with trisomy detected at gestational ages as early as 14th week.

Whole fetal genome mapping by parental haplotype analysis can be done by using sequencing of cell free fetal DNA.

mRNA transcripts from genes expressed in the placenta is detectable in maternal plasma.

Noninvasive prenatal paternity tests can be done as soon as nine weeks after conception.

The identification of fetal sex for women who are carriers of X-linked diseases is advantageous.

X-linked diseases occur 5 in 10,000 live births.

The most commonly sex-linked diseases include Duchenne muscular dystrophy and hemophilia.

Ultrasonography is unreliable during the first trimester of pregnancy, as the genitalia are not fully developed.

Other methods for sex determination before the use of cffDNA are invasive and performed at 11 weeks of gestation.

Most X-linked diseases are evident in males because they lack the second X-chromosome that can compensate for the diseases allele.

X-linked diseases include fragile-X syndrome, Duchenne muscular dystrophy and Hemophilia.

In the case of X-linked diseases, if cffDNA can determine gender, invasive testing can be eliminated.

Lack of the Y chromosome in the maternal plasma suggests that the fetus is female

Severe monogenic diseases for which prenatal diagnosis is applied include: cystic fibrosis, beta-thalassemia, sickle cell anemia, spinal muscular atrophy, myotonic dystrophy, fragile-X syndrome, Duchenne muscular dystrophy and Hemophilia.

Both autosomal dominant and recessive disorders have been detected noninvasively by analyzing paternally inherited DNA.

Mixing of fetal cells carrying paternal RhD antigens into maternal blood may result in the sensitization of an RhD-negative mother.

Rhesus blood group (D antigen) is used to determine the risk of hemolytic disease in the fetus.

In hemolytic disease, the maternal antibodies destroy RhD-positive fetal red blood cells, and this leads to lethality for the fetus.

A significant amount of blood can be exchanged between mother and infant during birth, CVS, amniocentesis and accidents.

50 defined antigens on the surface of red blood cells indicate Rhesus blood group.

RHD gene determines the Rhesus D status.

15% of Caucasian females, 3-5% of black African females and <3% of Asian females are RhD-negative.

Prophylactic treatment is recommended for all RhD-negative pregnant women to prevent isoimmunization in case of RhD incompatibility.

Amniocentesis still serves as the gold standard diagnostic tool for those women who require antenatal fetal blood genotyping, but it has been suggested that technology using cell-free fetal DNA may ultimately replace this invasive procedure.

Aneuploidy can be detected using non-invasive prenatal tests.

There is an increase in quantity of cffDNA in maternal plasma for fetal trisomy 13 and trisomy 21, and it is not elevated in fetal trisomy 18.

Sampling of cffDNA from maternal blood is estimated to have a sensitivity of between 96 and 100%, and a specificity between 94 and 100% for detecting Down syndrome.

Sampling for Down syndrome

can be performed at 10 weeks of gestational age.

Quantitation of cffDNA in maternal blood for preeclampsia reveals levels are fivefold more in preeclampsia pregnancies than normal, and can be used as a screening tool.

Cell-free fetal DNA can be used for whole genome sequencing, thus determining the complete DNA sequence of every gene of the baby.

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