Quantitative Fluorescent Polymerase Chain Reaction (QF-PCR)

In Rapid Prenatal Diagnosis of

Fetal Aneuploidies

Fetal Karyotype: The Gold Standard of Prenatal Diagnosis

Prenatal diagnosis for common chromosome aneuploidies (21, 18, 13 trisomy and sex chromosomes) in high risk pregnancies is usually performed by conventional cytogenetic analysis of chromosomes in metaphase (karyotype analysis).

The standard karyotype analysis can detect both numerical and structural abnormalities in all 23 chromosome pairs. And is performed by using in vitro culture of fetal nucleated cells retrieved by amniocentesis, chorion biopsy or fetal blood sampling.

Limitations of Karyotyping Specific to Prenatal Diagnosis

Limitations which apply particularly to prenatal diagnosis are:

�Difficulty in culturing of fetal tissues (amniotic fluid, chorionic villus and fetal blood).

�Maternal cell contamination (in case of fetal tissues) that makes subsequent analyses problematic.

Furthermore, it is labor intensive and turnaround time for karyotyping is more, which is

generally considered to be a psychological burden and results in late terminations

following a diagnosis.

New Techniques for Rapid Diagnosis of Fetal Chromosome Anomalies

Molecular techniques have been developed for rapid prenatal diagnosis of the most common chromosome disorders.

Fluorescent In Situ Hybridization (FISH)

Quantitative Fluorescent Polymerase Chain Reaction (QF-PCR)

Rapid Prenatal Diagnosis

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*Note: For more details please refer to our Medimail September 2008 issue

QF-PCR in Prenatal Diagnosis

�Quantitative fluorescence polymerase chain reaction (QF-PCR) is a molecular (DNA)-based test performed on uncultured fetal cells.

�The principle of QF-PCR technique is based on the presence of highly polymorphic short tandem repeats (STR) markers specific for eachchromosome.

�The number of alleles present in a sample can be determined by using fluorescent labeled primers for PCR amplification of STR markersspecific for each chromosome followed by quantitative analysis.

STRs are hyper-variable regions of the genome with repeat elements of 2-7 bases in length

Result Interpretation

Normal Sample: will show two peaks of similar fluorescent intensities and a ratio of 1:1 corresponding to the presence of two alleles at each specific STR locus.

Abnormal Sample: will produce either three STR peaks with ratios of fluorescent intensity of 1:1:1 corresponding to the presence of three different STR alleles, or two peaks with ratios of 2:1.

Maternal cell contamination: If two genotypes are seen within a sample, this indicates maternal cell contamination.

If a Down’s syndrome Fetus carries Translocation 21/21, it cannot be detected by QF:PCR

Advantages of QF-PCR in Prenatal Diagnosis

� Since, prenatal testing is carried out during early gestation, the major advantage of QF-PCR is rapid analysis.

� Maternal cell contamination can be detected by QF-PCR.

� Highly polymorphic microsatellite markers are amplified to perform the diagnosis; these can provide additional information of great clinical value such as:

� The origin of the extra chromosome in trisomic samples and the meiotic phase where non disjunction occurred.

� Determination of the zygosity independently of the chorionicity and the fetal sex in case of multiple pregnancy.

Study Data

Vincenzo Cirigliano et al. Prenat Diagn 2009; 29: 40–49

� QF-PCR test was applied on 43 000 clinical samples.

� The majority of prenatal samples were amniotic fluids (37 544) & CVS (4687); 178 fetal blood samples & 591 fetal tissues from aborted fetuses were also investigated.

� Most common indications for an invasive procedure were biochemical risk (32%) and advanced maternal age (30%).

� Samples were also tested by cytogenetic analysis and the results compared.

� Aneuploidies involving chromosomes 21, 18, 13, X and Y were detected with 100% specificity.

� Several cases of partial trisomies and mosaicism were also identified.

� Overall 95% of clinically relevant abnormalities were readily detected.

Conclusion: QF-PCR is a reliable method for the rapid detection of aneuploides.

Test Offered at SRL

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Amniotic fluid, CVS, Whole Blood

Rapid Prenatal Diagnosis by QF-PCR

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