Maternal Cell Contamination resulting in Discrepant Uncultured Amniocyte FISH and Long Term Culture Results Detected b

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) Cytogenetics, Newcastle Upon Tyne

2. Prenatal Diagnostic Methods 3 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

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

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

5. 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 Bloody – No white/foetal cells visible – Only male results given (can’t be sure if female are foetal or maternal) Newcastle In-House Protocol for Uncultured Amniocyte FISH Pellet condition is recorded according to the level of blood staining:

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 Cultures – slow growing – All male metaphases - 2% Female on FISH – QF-PCR showed Foetal geneotype

7. M P M P P M M P M P P M M P Case 1 Uncultured Amniocytes Case 1 Culture b

8. Case 2 Case 2 Pellet Condition - >90% 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

9. Case 2 Uncultured Amniocytes Case 2 Culture b M P 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

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?

12. Acknowledgements Alison Hammersley- Clinical Cytogeneticist Jerry Evans - Clinical Cytogeneticist Prenatal Section – Cytogenetics Newcastle

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