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RAPID PRENATAL DIAGNOSIS OF COMMON ANEUPLOIDIES USING NOVEL METHOD MICROFLUIDICS-FISH IN COMPARISON TO CONVENTIONAL KARYOTYPING AND FISH A. Pietrzyk ¹, J.Gronwald ¹, M.Rylow ¹, E. Studniak ¹, S. Zajaczek ¹ 1) Cytogenetic Unit, Pomeranian Medical University, Szczecin, Poland
INTRODUCTION
MATERIAL AND METHODS
RESULTS
Conventional karyotyping remains a golden standard in invasive prenatal diagnosis but with new molecular diagnostic procedures such as: FISH, QF-PCR or MLPA, implemented for rapid prenatal detection of specific chromosomal abnormalities, the choice of single effective method is being discussed. Microfluidics- FISH is a novel technique based on FISH, combining its different steps into a microchip, a special device with microchannels and glass coated with nanostructured titatnium dioxide. This integral approach allows to obtain result for common aneuploidies within the same day from a much smaller sample of amniotic fluid (1-4ml), being cost-effective, as it uses even 20 fold less reagents.
45 samples of amniotic fluid have been obtained from pregnant women between 15+² and 18+³ weeks of pregnancy, in which amniocenteses were performed due to specific indication: increased risk for aneuploidies in prenatal screening ( ultrasound scan and maternal serum markers). Cell cultures have been set up for conventional karyotyping. Concurrently, uncultured, Carnoy-fixed amniotic cells have been assessed examined for trisomy 21, 13, 18 and sex chromosomes numeric aberrations with FISH and Microfluidics- FISH methods, using commercially available kits of probes. The results obtained by means of standard karyotyping and both FISH methods have been compared.
Conventional karyotyping showed: normal fetal karyotype in 80% of analyzed cases and abnormal in 20% (including: trisomy 21 in 13,3%, trisomy 18 in 2,2%, triploidy in 2,2% and Turner syndrome in 2,2%). No other numerical or structural aberrations have been found. Both Microfluidics- FISH and FISH method showed 100% accordance with classical karyotyping. 90% of Microfuidics- FISH results has been obtained within 24 hours of amniocentesis, and accurate diagnosis of trisomy 18, triploidy and Turner syndrome was achieved in six hour tournaround time. Results of FISH were complete on the third day since sample drawing. Normal fetal karyotype Trisomy 21
AIM
Trisomy 18
The aim of this study was to compare diagnostic effectiveness and turnaround time for Microfluidics- FISH in comparison to conventional karyotype and FISH.
Normal karyotype of male fetus
Normal results of FISH Probes for centromere regions: - green : X chromosome - red: Y chromosome - aqua: 18 chromosome
and
Triploidy Monosomy X
Microfluidic- FISH
Microfluidic FISH Probes for: - green: 13q14 region - red: 21q22 region
Trisomy 18
Triploidy
Probes for centromere regions: - green : X chromosome - aqua: 18 chromosome
Probes for: - green: 13q14 region - red: 21q22 region
Trisomy 21
Monosomy X
Probes for: - green: 13q14 region - red: 21q22 region
Probes for centromere regions: - green : X chromosome - aqua: 18 chromosome
CONCLUSIONS For most cases turnaround time was shortest for MicrofluidicsFISH (1 day) and average volume of sample was smallest (3-4ml). Microfluidics- FISH proved to be cost-effective, rapid testing method of common aneuploidies. Recognizing limitations of methods based on FISH it cannot replace conventional karyotyping and be the sole method of diagnosis, but is a valuable screening test that allows to alleviate parental anxiety within the same day of amniocentesis.
INTRODUCTION
MATERIAL AND METHODS
RESULTS
Conventional karyotyping remains a golden standard in invasive prenatal diagnosis but with new molecular diagnostic procedures such as: FISH, QF-PCR or MLPA, implemented for rapid prenatal detection of specific chromosomal abnormalities, the choice of single effective method is being discussed. Microfluidics- FISH is a novel technique based on FISH, combining its different steps into a microchip, a special device with microchannels and glass coated with nanostructured titatnium dioxide. This integral approach allows to obtain result for common aneuploidies within the same day from a much smaller sample of amniotic fluid (1-4ml), being cost-effective, as it uses even 20 fold less reagents.
45 samples of amniotic fluid have been obtained from pregnant women between 15+² and 18+³ weeks of pregnancy, in which amniocenteses were performed due to specific indication: increased risk for aneuploidies in prenatal screening ( ultrasound scan and maternal serum markers). Cell cultures have been set up for conventional karyotyping. Concurrently, uncultured, Carnoy-fixed amniotic cells have been assessed examined for trisomy 21, 13, 18 and sex chromosomes numeric aberrations with FISH and Microfluidics- FISH methods, using commercially available kits of probes. The results obtained by means of standard karyotyping and both FISH methods have been compared.
Conventional karyotyping showed: normal fetal karyotype in 80% of analyzed cases and abnormal in 20% (including: trisomy 21 in 13,3%, trisomy 18 in 2,2%, triploidy in 2,2% and Turner syndrome in 2,2%). No other numerical or structural aberrations have been found. Both Microfluidics- FISH and FISH method showed 100% accordance with classical karyotyping. 90% of Microfuidics- FISH results has been obtained within 24 hours of amniocentesis, and accurate diagnosis of trisomy 18, triploidy and Turner syndrome was achieved in six hour tournaround time. Results of FISH were complete on the third day since sample drawing. Normal fetal karyotype Trisomy 21
AIM
Trisomy 18
The aim of this study was to compare diagnostic effectiveness and turnaround time for Microfluidics- FISH in comparison to conventional karyotype and FISH.
Normal karyotype of male fetus
Normal results of FISH Probes for centromere regions: - green : X chromosome - red: Y chromosome - aqua: 18 chromosome
and
Triploidy Monosomy X
Microfluidic- FISH
Microfluidic FISH Probes for: - green: 13q14 region - red: 21q22 region
Trisomy 18
Triploidy
Probes for centromere regions: - green : X chromosome - aqua: 18 chromosome
Probes for: - green: 13q14 region - red: 21q22 region
Trisomy 21
Monosomy X
Probes for: - green: 13q14 region - red: 21q22 region
Probes for centromere regions: - green : X chromosome - aqua: 18 chromosome
CONCLUSIONS For most cases turnaround time was shortest for MicrofluidicsFISH (1 day) and average volume of sample was smallest (3-4ml). Microfluidics- FISH proved to be cost-effective, rapid testing method of common aneuploidies. Recognizing limitations of methods based on FISH it cannot replace conventional karyotyping and be the sole method of diagnosis, but is a valuable screening test that allows to alleviate parental anxiety within the same day of amniocentesis.
