1. Maternal Cell Contamination resulting in
Discrepant Uncultured Amniocyte FISH
and
Long Term Culture Results
Detected by QF-PCR
I’m Karen Thompson, I’ve been an MTO in Newcastle Cytogenetics for 10yrs. Over the past year I’ve been working up the QF-PCR multiplex to replace our uncultured amniocyte FISH in the prenatal section
Karen Thompson
MTO 2 (Genetic Technologist)
Cytogenetics, Newcastle Upon Tyne

2. Prenatal Diagnostic Methods
Aneuploidy accounts for >90% of chromosomal abnormalities with birth defect
FISH – Rapid result for aneuploidy within 3 working days – 13, 18, 21 and the sex chromosomes. - can’t 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 – Can’t detect the sex chromosome aneuploidies such as Turner Syndrome, can’t 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
I’ll quickly explain the prenatal diagnostic methods
There are 3 common methods used in prenatal diagnosis, two of which give rapid results but chromosome information is limited, and one which takes longer but can give more information about the karyotype. Aneuploidy of chromosomes 13,18, 21 and the sex chromosomes account for 90% of chromosome abnormalities and these are detected very quickly
FISH on uncultured amniocytes can give a rapid aneuploidy result for chromosomes 13, 18, 21 and the sex chromosomes, usually within 2 days. But this method can’t detect other chromosome aneuploidies or abnormalities and blood stained samples can be problematic because there is no way of telling if the signal pattern is from maternal cells or foetal cells.
QF-PCR is another 2 day method for detecting aneuploidy of chromosomes 13, 18 and 21. A separate sex multiplex is used to detect the sex chromosome aneuploidies but very few labs do this.A smaller volume of amniotic fliud is needed, so this leaves more cells for the cultures. MCC is problematic with this method but it is less labour intensive and more cost effective than FISH and is capable of a higher throughput.
Long term cultures are more labour intensive than the other 2 methods, taking about two weeks for a result, but more chromosome information is available and MCC is not usually a problem because maternal cells are not spontaneously dividing.

3. 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
1.   So because QF-PCR is a more cost effective method, we’ve been trialling this alongside FISH so that we can make the change over.
We’ve trialled almost 500 AF samples alongside the FISH.
Routinely we take 4ml of AF for FISH and if there’s enough, 1ml for QF-PCR
While running the trial, it came to light that we may have a problem with our in house guidelines for analysing uncultured amniocyte FISH because we’ve had 2 discrepant results between FISH and long term culture results.

4. 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
1.     There are ACC guidelines for QF-PCR analysis.
This states that it is acceptable to process all blood stained samples.
But if the maternal genotype is present at a high level or if the allele ratios are inconclusive, that is the ratios fall outside the normal range, then the foetal geneotype must not be interpreted.
However, there are no such guidelines for uncultured amniocyte FISH analysis.
So, in Newcastle, we have our own in house guidelines.

5. Newcastle In-House Protocol for Uncultured Amniocyte FISH
Pellet condition is recorded according to the level of blood staining:
Clear - no blood visible – 50 cells scored
Trace - < 50% blood staining – 50 cells scored
Plus - > 50% blood staining but white/foetal cells still visible – 100 cells scored
1.     These guidelines came about after one of our trainees did data analyses on FISH results from blood stained samples.
This involves recording the pellet condition to the level of blood staining and scoring a minimum number of cells accordingly. The 4 categories are clear, trace, plus and bloody.
A clear pellet has no blood staining at all therefore 2 people score 25 cells each.
A pellet with less than 50% blood staining is classified as a trace, and again 2 people score 25 cells each.
A pellet characterised as plus, has more than 50% blood staining but white cells and foetal cells are still visible. Here 2 people score 50 cells each, or enough to score 25 male cells each.
The same amount is scored for a bloody pellet when foetal cells are not visible. In these cases we do not report a female result as we can not determine if this is foetal or maternal.
Bloody – No white/foetal cells visible – Only male results given (can’t be sure if female are foetal or maternal)

6. Case 1 Pellet Condition - unusually large, < 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
1.     So I’ll explain the 2 discrepant cases.
The pellet from the first case was unusually large with less than 10% blood staining and this was classified as a trace.
According to our in house guidelines 2 people scored 25 cells each and both agreed it was a female signal pattern.
QF-PCR showed evidence of a second genotype, which could have been the maternal genotype since there was a small trace of blood.
But we use the AMEL marker in our multiplex. This is a non polymorphic marker which has an X allele at about 106bp and a Y allele at about 111bp. So a normal female and a Turners would have only 1 peak at 106bp and a male pattern would show an X peak and a Y peak in a 1:1 ratio, except in very rare cases where the Y allele is polymorphic so it doesn’t give a Y peak.
So in this case there is an X allele and a Y allele but no where near a 1:1 ratio. You can see that the Y allele is much smaller in height and area than the X allele. These arrows point to evidence of a second genotype on some of the other markers.
So we scored more cells on the FISH slide and found 2 male cells out of 150.
We did QF-PCR on maternal blood and this matched the pattern on the uncultured amniocyte QF-PCR, except for the Y allele and the second geneotype.
The cultures were very slow growing. But after 17 days when they were eventually ready for harvesting, the metaphases were all male, FISH showed 2% female signal pattern and QF-PCR showed the foetal genotype was predominant.
Cultures – slow growing – All male metaphases - 2% Female on FISH – QF-PCR showed Foetal geneotype

7. Case 1 Uncultured Amniocytes
Case 1 Culture b M P
1.     So here are the QF-PCR plots for comparison.
In the uncultured amniocyte plot you can see evidence of a second genotype, which could have been interpreted as the maternal geneotype if we excluded the AMEL marker. But because the Y allele is comparatively small, alarm bells were ringing.
This is the foetal genotype from the culture. You can see that the X and Y alleles are more of a 1:1 ratio and many of the alleles are shared with the mother. These peaks under the arrows on the uncultured plot coincide with the paternal alleles on the plots of the foetus from the culture. This hump on D13S628 on the culture could be the other maternal allele but it could also be stutter.
So, we thought this was a one off and we were very lucky to have found it, but a few weeks later, case 2 crops up.

8. Case 2 Case 2 Pellet Condition - >90% blood staining
FISH on Uncultured Amniocytes 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
This time the pellet was a standard size but there was about 90% blood staining, so this was classified as a plus.
According to in house guidelines, 2 people scored 50 cells each on FISH and this gave a female signal pattern. Because this was a pus and not a bloody pellet, we would have reported it if we hadn’t done QF-PCR
Because the QF-PCR showed evidence of a second genotype and a Y allele.
Again the Y allele was very small compared to the X allele. In fact if you exclude the AMEL marker you could argue that there is only one genotype here as these two peaks could be stutter and not a second genotype.
Again we rescored the FISH and found 3 male cells out of 170.
QF-PCR on maternal blood confirmed that the predominat geneotype of the uncultured amniocytes was maternal.
Again cultures were very slow growing and G-band analysis showed only female metaphases in culture A with about 6% of male cells on FISH and maternal geneotype on QF-PCR.
But culture B had only male metaphases and QF-PCR showed the foetal genotype.
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

9. Case 2 Uncultured Amniocytes
Case 2 Culture b
1.     So here are the markers from the uncultured amniocytes and from culture B.
So you can see that the X and Y alleles are in a 1:1 ratio. The foetus is homozygous for D13S628. In D13S634, this allele at 414bp is from mum and this other one is from dad.
And in this marker, this allele at 290bp is from mum and this one at 278 is from dad.
These alleles in D18S1002 are identical to the maternal alleles.
This allele here at 209bp is from mum and if we look at the uncultured amniocyte plot we can see evidence of the paternal allele at 220bp, which is this one here. P M M P M P

10. 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 > 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
1.     So to Summarise
In a short space of time we’ve had 2 cases showing discrepant results with FISH and long term cultures and these were detected by QF-PCR.
The AMEL marker used in the QF-PCR multiplex suggested that the second genotype was foetal in both cases and the predominant genotypes were maternal.
The first case had an unusually large cell pellet with only a trace of blood but still gave a female signal pattern on FISH however, the male foetal cells prevailed in culture.
Case two on the other hand was 90% blood stained, with a female signal pattern on FISH, but one culture was predominantly maternal and the other culture was predominantly foetal.
Cultures from both cases were very slow growing and this was possibly due to the high level of non spontaneously dividing maternal cells.
The professional guidelines for QF PCR would have prevented us from giving a maternal result rather than the foetal result, at least in case 1
However, there are no such guidelines for FISH, therefore according to our in house guidelines we would have reported the two rapid FISH results.
QF_PCR was useful to determine the origin of the cultures.

11. 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?
1.     So, in conclusion,
Seeing only a trace of blood in the cell pellet does not mean that the result will be foetal
This is particularly important when the cultures are slow growing or the cell pellets are unusually large.
QF-PCR is useful to determine the origin of female cultures when maternal blood is available.
In male cases, the AMEL marker is useful to estimate the level of MCC
Also, these two cases have made us re think our protocol for analysing and reporting uncultured amniocyte FISH. Maybe there is a need for professional guidelines.

12. Acknowledgements Alison Hammersley- Clinical Cytogeneticist
Jerry Evans - Clinical Cytogeneticist
Prenatal Section – Cytogenetics Newcastle
I’d like to thank Alison Hammersly who has supervised the QF-PCR set up
Jerry Evans, the head of the prenatal section
And not forgetting the rest of the prenatal section for covering my duties while I’ve been working on the PCR set up

13. Maternal Cell Contamination resulting in
Discrepant Uncultured Amniocyte FISH
and
Long Term Culture Results
Karen Thompson
MTO 2 (Genetic Technologist)
Cytogenetics, Newcastle Upon Tyne