C- cell free DNA

overview

define cell free DNA

short (<200bp) dsDNA fragments that circulate freely outside of cells in bodily fluids (plasma and urine) and represent the entire genome

in pregnancy, a portion of cfDNA in maternal plasma comes from placenta = cell-free fetal DNA (cffDNA)

cffDNA becomes undetectable a few hours post partum and is not influenced by previous pregnancies

origin of cffDNA in maternal circulation

enters through placental trophoblast turnover

the cells are rapidly destroyed by maternal immune system → releasing DNA fragments

define fetal fraction

= proportion of fetal cfDNA relative to total cfDNA

minimum FF of 4% required for reliable analysis

• low FF increases false positives and nocall results

average FF= about 10% at 11-14 weeks gestation

FF is affected by :

• gestational age

• placental size

• fetal aneuploidy (higher in T21, lower in T13/T18)

• maternal obesity reduces FF due to increased maternal cfDNA (from adipocyte turnover)

clinical purpose of cfDNA testing

prefer to call it maternal plasma cfDNA screening

screening test that assesses risk for

• trisomy 21,18 and 13

• sex chromosome aneuploidies

results must be confirmed with invasive diagnostic testing before clinical decisions are made

when is cfDNA screening funded

for high risk pregnancies under 2 categories

• category I = can be ordered by any physician or NP

• category II = must be ordered by genetics of maternal-fetal medicine specialists and requires pre and post-test counseling

microdeletion screening is not publicly funded and is not routinely recommended

sample collection and pre-analytical considerations

non invasive (refering to safe for fetus) → just a blood draw

pre analytical concerns

• prevent WBC lysis (releases maternal gDNA → diluting FF)

• use special tubes to prevent this

• rapid processing needed

• temp control during transport

reasons for sample rejection

• insuficient gestational age

• incorrect tube or labeling

• hemolysis, insufficient volume, freezing, or delayed receipt

sample preocessing

double centrifugation

• low speed to separate plasma

• high speed to remove residual cellular material

use extraction kits optimized for short fragments

• often magnetic beads or membrane binding

• often automation to reduce contamination risk

analytical mehods

counting-based methods

• using NGS, millions of cfDNA fragments are seq and mapped

• the number of reads per chrom is compared to expected values

• relative excess/deficit → aneuploidy

  • shotgun MPS = seq fragments from all chroms

  • targeted MPS = focuses on selected chroms to reduce cost and improve efficiency

• cannot distinguish maternal from fetal DNA

SNP-based genotyping

• amplifies 20k SNPs and compares maternal and fetal alleles

• differences in ratios and clustering patterns allows estimation of fetal fraction and detection of aneuploidy

• need advanced algorithms not visually obvious results

performance of cfDNA screening

high sensitivity and specificity for trisomy 21

slightly lower performance for T13 and T18

false positives and more common than false negatives

false positive may come from

• CPM

• vanishing twin syndrome

• maternal chromosomal abn or malignancy

• prior organ or bone marrow transplant

false negatives often come from low FF

results and follow up

reported as High or Low risk

within 4-10 days

failed te due to low FF or tech issue and is associated with increased aneuploidy risk

positive screens must be confirmed by diagnostic testing