Genomics OSFA 2019 Flashcards
Describe culturing of chorionic villi samples/amniotic fluid samples
Which steps do we take to protect from errors?
Aseptic techniques
Class II hood that protects the sample and the user
Blood and maternal decidua removed
Villi enzymatically digested (mixing several villi reduces the risk of false positives/negatives due to CPM)
Blood stained amniotic fluids: Centrifugate through gradient to separate amniocytes and RBCs
2-3 cultures are set up in different incubators, so that if a culture is lost due to contamination/incubator fault, back-ups are available and culturing artefacts can be verified/ruled out from a second culture
Infection:
Culture medium contains antibiotics
Change of pH can be a sign of infection (bacteria (pink->yellow), fungi (pink->red), not so much yeasts)
Cloudiness (generally bacterial)
Granular, shimmering appearance (=bacteria), particles, sometimes chains (=yeast), mycelia filaments (fungi) can be seen in x100 phase contrast
When single or multiple abnormal cells are found in CVS or AF cultures, more cells can be analysed. Guidelines describe basic, moderate and extensive workup with a number of cells from 1 or more separate cultures to exclude culturing artefacts.
QF-PCR trisomy result from a CVS shows only biallelic markers - what to do next and why?
Use back-up markers to try and get a tri-allelic result
A bi-allelic result may result from a mitotic event in the placenta and represent confined placental mosaicism which is not representative of the fetus
Confirm the ID with maternal sample (if not available: Confirm result by FISH)
QF-PCR on a CVS or amniotoc fluid shows 1 marker with skewed ratio between 2 peaks or a 3rd allele with lower peak height. What can be done and what may this signify?
Repeat marker at 53 and 58 degrees: Marker may resolve to normal ratio
Check if marker is flanked by normal markers
Run parental sample if available
A SNP under primer binding site may have reduced the primer efficiency producing a peak with lower height
If inconclusive ratio or three alleles persists this could be due to microsatellite instability or submicroscopic duplication
What to do if the QF-PCR result for a prenatal sample shows maternal cell contamination?
In cases without abnormal scan results: Do karyotyping on cultured cells
In cases with abnormal scan results: Do microarray on cultured cells
Culturing may result in amniocytes/chorionic vili digest to outgrow the leucocytes and thereby overcome the MCC
Contact the clinician to inform them of the delay to the result
A QF-PCR result on a CVS shows evidence of trisomy 21 in 3/5 markers (1 biallelic and 2 triallelic). What to do next?
What if it was an amniotic fluid?
CVS: Use the maternal sample for sample ID check to ensure there has been no sample mix-up in the laboratory
Amniotic fluid: Extract from a banked aliquot or a pour-off from the culture and repeat the test to confirm sample ID
Report as a preliminary result and activate long-term cultures for karyotyping to confirm the mode of trisomy (regular/Robertsonian or other translocation)
If translocation involved request parental samples to determine if inherited or de novo to determine recurrence risk
A QF-PCR result on a CVS shows evidence of monosomy X. What to do next?
What if it was an amniotic fluid?
Repeat the XY QF-PCR
CVS: Confirm the ID with maternal sample
Amniotic fluid: Extract from a banked aliquot or a pour-off from the culture and repeat the test to confirm sample ID
A XXY result was seen by QF-PCR on an amniotic fluid. The sample was referred due to choroid plexus cysts and duodenal atresia.
What do you do next?
Repeat the QF-PCR on new extraction to confirm the result and sample ID
Look at microarray result to see if this confirms the finding (if this was not an abscan, findings from QF-PCR that does not explain the referral reason could still be followed up by microarray).
Could do karyotyping if suspect mosaicism and would like an estimate of level.
Word the report very carefully to highlight that this is an incidental finding, which does not explain the scan findings
Explain the significance of Klinefelter disease
Mosaic trisomy 2 was seen on a microarray on a CVS sample.
What do you do next?
Consider if this is a true or false result
Repeat the test on new extraction (cultured cells) for ID check or do FISH (if recurrent submicroscopic imbalance) or karyotype (can estimate the level of mosaicism)
Could it be due to CPM?
If CPM then repeat test on long-term culture or take an amniotic fluid at a later date, where CPM is not a problem
Is mosaic chromosome 2 compatible with life?
A CVS sample arrives with referral due to raised NT and raised risk from combined test (1 in 5). Gestation by scan is 13+0 weeks.
What would you do when triaging this sample?
Clarify with clinician if the raised NT is >3.5 mm to determine if this is an abnormal scan finding and needs microarray.
If NT <3.5 mm:
Do QF-PCR on CVS digest (if there is enough material for digest)
Bank a long-term culture
Extract DNA from maternal sample for MCC studies
If NT >3.5 mm:
All of the above and also initiate microarray analysis and XY QF-PCR
Explain QF-PCR
Quantitatively amplify regions of DNA that include tetranucleotide microsatellite markers, which are polymorphic in population. (Tetranucleotide markers are less prone to polymerase slippage than smaller repeats, therefore smaller stutter peaks). Not too many cycles, as want to capture exponential phase. Analysed using fluorescent capillary electrophoresis. Five markers on each chromosome 13, 18 and 21 are analysed. AMEL marker is not polymorphic, but always 103 bp on X and 109 bp on Y). Back-up kits with 4 extra markers are available if first kit only has uninformative markers or are all bi-allelic.
Ratio of 0.8-1.4 (1.5 further apart) is normal. Ratio of <0.65 or >1.8 is trisomic.
If only 1 allele is seen, this could be due to drop-out of other allele of patient is homozygous at this locus (uninformative).
Skewing of AMEL markers may indicate sex chromosome aneuploidy or contamination (e.g. MCC).
If 1 marker only gives a result that conflicts with other markers: Repeat single marker. Can lower annealing temperature (if polymorphism is present under primer binding site). Can do back-up markers if surrounding markers are uninformative. Tri-allelic result in 1 marker only may be true result from somatic instability (MSI) or submicroscopic duplication (SMD).
Run parental samples: Can see if SMD inherited.
Which microarray findings are reported for prenatal samples?
Always reported:
Findings related to scan abnormalities
Pathogenic findings unrelated to scan findings, but with near full penetrance (likely to cause phenotypic effect, actionable incidental findings)
High penetrance neuro-susceptibility loci associated with severe phenotype
Not reported:
Benign, likely benign or uncertain significance findings
Low penetrance findings (susceptibility loci), which do not include features detectable on scan
Describe the antenatal screening programme (NHS Fetal Anomaly Screening Programme (FASP))
First trimester combined screen (/NT screen): Maternal serum markers (free ß-human chorionic
gonadotrophin (hCG) and pregnancy-associated plasmaprotein-A (PAPP-A)), nuchal translucency and maternal age combined into risk. Offer CVS if high risk (>1:150).
If women book late they can have the Quadruple test: Maternal serum markers (alpha-fetoprotein (AFP),
free ß-human chorionic gonadotrophin (hCG), unconjugated estriol (uE3) and inhibin-A) and maternal age are combined into risk. Offer amniocentesis if high risk (>1:150).
Testing done by QF-PCR, which can detect trisomy 21, 18 and 13, triploidy and monosomy X. They are screened for because they are relatively common and can result in abnormal scan findings, confer a risk of spontaneous abortion or stillbirth and may lead to live born children with mental and physical problems. The test may detect other abnormalities (other sex chromosome abnormalities, mosaic forms or unbalanced translocations).
If QF-PCR is positive: Do karyotyping.
If QF-PCR is negative: Do microarray
What to put in report for a prenatal QF-PCR result showing a trisomy?
What if it was bi-allelic only also in back-up markers?
Provisional result Indicates trisomy Sample identity confirmed Fetal sex No evidence of other trisomies CPM can occur (if CVS, 2% of viable pregnancies) Confirmation by karyotype will follow List features of trisomy
If bi-allelic markers only word more carefully to indicate that could be CPM
When can a CVS be taken?
When can an amniotic fluid be taken?
When can maternal sample be taken for NIPT?
CVS: Usually between 11-13 weeks (must not be performed before 10 weeks)
AF: Usually after 15 weeks (must not be performed before 14 weeks)
NIPT: cffDNA can be detected from 7 weeks, but blood sample not taken until 10 weeks
In which circumstances can NIPT be offered?
In which circumstances can CVS/AF be offered?
NIPT: Following high risk screening for T21, 13, 18
If abnormalities seen on ultrasound scan then CVS or AF may be better as may need microarray
CVS/AF: NIPT high risk Screening high risk (NIPT declined) Previous pregnancy aneuploidy Known familial translocation (e.g. Robertsonian)
