20.05.01 Prenatal sampling and considerations Flashcards
Why do prenatal testing?
We offer screening and diagnostic tests to help make informed choices about: 1) pregnancy management 2) termination 3) outcomes 4) recurrence risk
Types of prenatal samples
1) cffDNA
2) CVS
3) Amnio
4) fetal blood sampling - can also test pregnancy loss samples (placenta, POC) , fetal skin/fibroblasts
cffDNA
- fetal DNA circulating in mat blood (2-6% of total DNA)
- Originates from apoptotic trophoblasts in placenta
- Present from 5 weeks (not enough to sample until 10 weeks)
- 200bp in size (significantly smaller than maternal fragments)
CVS
- placental tissue made up of trophoblasts and mesenchyme cells
- remove 10-25mg of CV
- performed transabdominally or transcervically via a needed and syringe under US guidance
- 1-2% risk of miscarriage (can cause infection, leakage of AF, or damage to placenta)
- NHS offers CVS from 11+0 to 13+6 weeks gestation
- 1.5-2% of samples have CPM, and MCC also an issue
- Maternal deciduas is removed by lab to reduce MCC risk
- Pro = earlier diagnosis
- Con = slightly higher miscarriage rate and CPM/MCC
Amniocentesis
- Fluid filled sac = amnion
- Required to maintain body temp, aide symmetrical growth and lung development
- Take <20mls transabdominally using a syringe and hollow needed by US
- 0.5-1% risk of miscarriage (can cause infection, or placental/fetal damage)
- Carried out from 15 weeks
- AF cell population is heterogeneous (25% is fetal tissue from lungs, urinary tract, skins, 10% extra-embryonic membranes, and 65% amniocytes)
- Gives a more accurate view of fetal karyotype than CVS
- Pro = lower risk of miscarriage and more accurate for fetal genotype
- Con - Later diagnosis
Fetal blood sampling (FBS)
- Taken from blood vessels of umbilical cord or fetal blood vessel
- After 18-20 weeks gestation when veins are developed
- Insertion of needle into umbilical cord under US
- Reliable indicator of fetal genotype
- Higher risk of fetal loss (2%)
- Only used after inconclusive results
- Can be used for mTOP
MCC
- level fo MCC linked to sampling technique and operator
- CVS - risk of maternal decidua (higher risk)
- Amnio - risk from blood staining
- Tested for using polymorphic microsatellite markers (QF-PCR) by comparing mat blood to CVS/AF
MCC and culturing techniques
- AF - culturing REDUCES risk of MCC because culture favours growth of amniocytes and reduces/eliminates mat blood cells
- CVS/POC - culturing INCREASES risk of MCC given the co-localization of mat and fetal cell lineages
- Always have back up cultures if uncultured sample shows MCC
- Bloodstained early gestation AF - higher MCC risk (as early gestation amnios have fewer amniocytes than later gestation AF samples)
What are the BPG for when to suspect MCC?
1) Mix of XX and XY cells
2) Female abnormal and female normal cells
3) Female karyotype is different to previous diagnosis/fetal sex
4) Unsure about identity of sample in culture
5) Slow growing samples
When to carry out MCC testing?
1) Molecular prenatal tests
2) All testing done on FBS, cord blood and POC samples
- Min of 2 markers needed to sig rule out MCC
- Test needs to pick up >10% level of contamination
Prenatal mosaicism in AF samples
- occurs in 0.1-0.3% of AF samples
- 3 levels of mosaicism:
1) single abnormal cell - pseudomosaicism
2) 2 or more identical abnormal cells seen in a single culture - additional studies can be performed but almost always pseudomosaicism
3) 2 or more identical abnormal cells seen in 2 or more independent cultures - likely reflects true fetal mosaicism
Confined placental mosaicism
- abnormal cells restricted to extra-embryonic tissues
- detected in 1-2% of pregnancies at 10-12 weeks in CVS
- 80% of autosomy trisomy mosaicism in CVS is CPM
1) 50% confined to trophoblasts
2) 30% confined to villus mesenchyme
3) 20% confined to trophoblast and mesenchyme - Most cases are trisomy in placenta, diploid in fetus
- 10% of cases fetus is abnormal
- CPM should be suspected when QFPCR only shows trisomy with bi-allelic peaks only (and NO tri-allelic peaks)
- Can be numerical (most common) or structural
2 molecular mechanisms for CPM
1) Mitotic CPM - Mitotic non-disjunction occurs in trophoblast or non-fetal cell line in ICM
- Creates trisomic cell line in tissue which becomes placental mesoderm
- Trisomy 2, 3, 7 and 8 occur this way
2) Meiotic CPM - Trisomy conception undergoes trisomic rescue in some cells (including those that will become the fetus)
- Remaining trisomy cells can be confined to the placenta
- Trisomy 16 and 22 occur this way
What factors influence the pattern of normal and abnormal cells in the developing embryo?
1) Reduced or increased replication rate in trisomic cells compared to normal cells
2) Abnormal cells may fail to differentiate/function
3) Presence of abnormal cells may compromise pregnancy (IUGR, IUD etc)
Three types of CPM
- CVS usually contains a mixture of cytotrophoblasts and mesenchymal cells
- Direct preparation and short term cultures are mostly cytotrophoblasts and long term cultures and mostly mesenchymal cells
- 3 types of CPM:
1) abnormal cells confined to cytotrophoblast (40%)
2) abnormal cells confined to mesenchymal cells (40%)
3) abnormal cells present in both tissues (7%) - Types 1 and 2 due to nondisjunction events
- Type 3 due to trisomy rescue (at increased risk of complications and UPD)
- trisomies 13, 18 or 21 most likely to be fetal in origin
- trisomies 8, 9, 12, 13, 15, 18, 20, 21, and sex chr usually involve the fetus and are high risk for true fetal mosaicism
