Microfluidic digital PCR enables rapid prenatal diagnosis of fetal aneuploidy

Abstract

Objective: The purpose of this study was to demonstrate that digital polymerase chain reaction (PCR) enables rapid, allele independent molecular detection of fetal aneuploidy.

Study design: Twenty-four amniocentesis and 16 chorionic villus samples were used for microfluidic digital PCR analysis. Three thousand and sixty PCR reactions were performed for each of the target chromosomes (X, Y, 13, 18, and 21), and the number of single molecule amplifications was compared to a reference. The difference between target and reference chromosome counts was used to determine the ploidy of each of the target chromosomes.

Results: Digital PCR accurately identified all cases of fetal trisomy (3 cases of trisomy 21, 3 cases of trisomy 18, and 2 cases of triosmy 13) in the 40 specimens analyzed. The remaining specimens were determined to have normal ploidy for the chromosomes tested.

Conclusion: Microfluidic digital PCR allows detection of fetal chromosomal aneuploidy utilizing uncultured amniocytes and chorionic villus tissue in less than 6 hours.

FIGURE 1. Sample false-color images of microfluidic digital PCR chips

These images are produced by overlaying the subtracted images in both fluorescent channels. FAM signal is shown in green, and HEX signal is shown in red. A red square represents a compartment containing amplification products giving out signal in the HEX channel (chromosome 1 locus). A green square represents a compartment containing amplification products giving out signal in the FAM channel (chromosomes X, Y, or 21 loci, as labeled on the sides of the images). A yellow square is an overlap of a red and a green square. A, Normal female fetus (46 XX). The number of green squares is comparable to that of red squares in panels targeting chromosomes 21 and X. No green squares are present in panels targeting chromosome Y. B, Male fetus with trisomy 21 (47 XY +21). The number of green squares is approximately half that of red squares in panels targeting chromosomes X and Y. More than expected number of green squares is observed in panels targeting chromosome 21. Comparison of green and red square counts reveals a ratio of approximately 3:2.

FIGURE 2. Digital PCR results

For each sample, the difference between target and reference chromosome counts is plotted against the reference chromosome count. The boundaries represent 99.9% confidence interval of each cases of ploidy. A, Chromosome 13 as the target chromosome. All but 2 samples fell within the region of disomy. Two cases of trisomy 13 were detected. B, Chromosome 18 as the target chromosome. Three cases of trisomy 18 were detected. The rest were determined to be normal. C, Chromosome 21 as the target chromosome. Three cases of trisomy 21 were detected. The rest were determined to be normal. D, Chromosome X as the target chromosome. All female samples fell within the region of disomy, while all male samples lied within the region of monosomy. E, Chromosome Y as the target chromosome. All male samples fell within the region of monosomy. None of the female samples showed amplification for chromosome Y assay.