Maternal Cell Contamination

1
Maternal Cell Contamination resulting
in Discrepant Uncultured Amniocyte FISH and
Long Term Culture Results Detected by QF-PCR
Karen Thompson MTO 2 (Genetic Technologist) Cytoge
netics, Newcastle Upon Tyne
2
Prenatal Diagnostic Methods
3 Methods Aneuploidy accounts for gt90 of
chromosomal abnormalities with birth defect FISH
Rapid result for aneuploidy within 3 working
days 13, 18, 21 and the sex chromosomes. -
cant detect translocations or deletions Blood
staining can be problematic QF-PCR Rapid result
for aneuploidy within 3 working days More cost
effective than FISH, higher throughput Cant
detect the sex chromosome aneuploidies such as
Turner Syndrome, cant detect translocations or
deletions Less amniotic fluid required -
Maternal cell contamination is problematic Long
term cultures two weeks for result more
labour intensive than other two methods detects
any chromosome abnormality within the resolution
of the microscope maternal cell contamination
not usually a problem
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Prenatal QF-PCR Trial in Newcastle
Approx 500 amniotic fluid samples alongside
uncultured amniocyte FISH
Routine FISH, QF-PCR when enough for a run
Two Discrepant amniocyte FISH and QF-PCR results.
Long term culture results also discrepant with
FISH
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Guidelines
QF- PCR ACC Best Practice Guidelines -
acceptable to process all blood stained samples
If the maternal genotype is present at a high
level and/or if the allele ratios are
inconclusive then the foetal genotype must not be
interpreted
Uncultured Amniocyte FISH No Professional
guidelines In house protocol only
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Newcastle In-House Protocol for Uncultured
Amniocyte FISH
Pellet condition is recorded according to the
level of blood staining
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Case 1
Pellet Condition - unusually large, lt 10 blood
staining
FISH on Uncultured Amniocytes - 50 cells scored
according to in house guidelines female signal
pattern
QF-PCR - Second genotype seen on some markers
very small compared to main genotype
Y allele using the AMEL marker skewed ratio
Uncultured amniocyte FISH re-scored 2 male/150
QF-PCR Maternal Blood Showed uncultured
amniocyte QF-PCR result was predominantly
maternal second genotype was foetal
Cultures slow growing All male metaphases -
2 Female on FISH QF-PCR showed Foetal
geneotype
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Case 1 Uncultured Amniocytes
Case 1 Culture b
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Case 2
Pellet Condition - gt90 blood staining
FISH on Uncultured Amniocytes - 100 cells scored
according to in house guidelines female signal
pattern
QF-PCR - Second genotype seen on some markers
very small compared to main genotype Y allele
using the AMEL marker skewed ratio
Uncultured amniocyte FISH re-scored 3 male/170
QF-PCR Maternal Blood Showed uncultured
amniocyte QF-PCR result was predominantly
maternal second genotype was foetal
Cultures slow growing
Culture a - All female metaphases 5.7 male on
FISH QF-PCR showed maternal genotype
Culture b - All male metaphases QF-PCR showed
foetal genotype
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Case 2 Uncultured Amniocytes
Case 2 Culture b
M
P
M
P
M
P
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Summary
Two cases showing discrepant FISH and Long term
culture results and this was detected by
QF-PCR AMEL marker - uncultured amniocytes
predominantly female in both cases Case 1
Unusually large cell pellet with a trace of blood
- foetal cells prevailed in culture Case 2
Standard size cell pellet with gt 90 blood -
maternal cells prevailed in culture a, but foetal
cells prevailed in culture b! Both cultures were
very slow growing - ?due to high level of
MCC Professional guidelines for QF-PCR
Therefore rapid result would have been
unacceptable No such guidelines for FISH
Therefore maternal result instead of foetal
result QF-PCR useful to determine origin of
cultures
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Conclusion
Identifying only a trace of blood, does not
equate to the genotype being predominantly
foetal Caution with results from unusually large
cell pellets and slow growing cultures QF-PCR is
useful to determine origin of cultures maternal
blood may be necessary in cases where slow
growing cultures result in a female karyotype In
male cases the AMEL marker is useful in QF-PCR
multiplex to show the level of MCC in both rapid
and long term diagnoses Revise in house protocol
for analysing and reporting FISH Professional
guidelines required?
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Acknowledgements
Alison Hammersley- Clinical Cytogeneticist Jerry
Evans - Clinical Cytogeneticist Prenatal Section
Cytogenetics Newcastle
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Maternal Cell Contamination resulting
in Discrepant Uncultured Amniocyte FISH and
Long Term Culture Results
Karen Thompson MTO 2 (Genetic Technologist) Cytoge
netics, Newcastle Upon Tyne