Evaluation of Microfluidics-FISH method in prenatal diagnosis

Abstract

Objectives: Classical cytogenetic analysis remains a gold standard in invasive prenatal diagnosis. Recently, Microfluidics--FISH, a novel method based on FISH, has been introduced. This integral approach allows to obtain result for common aneuploidies within the same day from a much smaller sample of the amniotic fluid. In this study we compare effectiveness of Microfluidics-FISH to classical karyotype and Rapid FISH.Material and methods: 52 samples of amniotic fluid were drawn from the pregnant women due to common indications. Cell cultures have been set up for classical cytogenetic analysis as well as amniotic cells have been loaded into the microchip of Microfluidics-FISH as well standard procedure of Rapid FISH was performed for evaluation of trisomy 21, 13, 18 chromosome and sex chromosomes numeric aberrations.Results: 9 samples out of 52 showed chromosomal aberrations in both FISH methods what was consistent with karyotyping. One case of small supernumerary marker chromosome was detected only in the classical cytogenetic analysis. For the majority of cases turnaround time was shortest for Microfluidics-FISH and the average volume of sample was smallest. Microfluidics-FISH proved to be reliable and cost-effective rapid testing method of common aneuploidies. Recognizing, however, limitations of methods based on FISH it cannot replace conventional karyotyping and be the sole method of diagnosis.

A novel use for Levey-Jennings charts in prenatal molecular diagnosis

“…We used FISH as a representative method to study the feasibility of establishing Levey-Jennings chart-based IQCs for monitoring molecular diagnostic quality, because FISH is widely used to detect aneuploidies after amniocentesis and to perform chromosome analysis of leukemic blood samples and other tissues [20][21][22][23][24]. In general, each clinical sample only includes one karyotype.…”

“…Thus, considering that the accuracy of aneuploidy detection by FISH can be improved by increasing cell counts [20][21][22]27], and that quality-control cells containing different proportions of karyotypes can more accurately assess detection quality [26], we used 60 and 20% as the thresholds for aneuploidy detection [28,29]. We prepared high-, medium-, and low-value qualitycontrol cells (A-H) with aneuploidy ratios of 10, 30, 70, and 90%, respectively, to provide a relatively consistent ratio of chimeras to the common clinical samples with various karyotypes as the basis to establish an ideal IQC for prenatal molecular diagnosis by FISH [20][21][22][23][24]. Although the quality-control cells transfected with SV40LT are not identical to primary amniocytes and Phaseolus vulgaris-leucoagglutinin (PHA)-stimulated lymphocytes used for FISH quality control approaches in 26 laboratories [30], they have several advantages as IQCs.…”

“…Compared with non-chimeric karyotypes, chimeric karyotypes are more common and are more easily misdiagnosed in prenatal diagnosis by FISH, particularly in individuals with a low proportion of multiple karyotypes [ 24 – 26 ]. Thus, considering that the accuracy of aneuploidy detection by FISH can be improved by increasing cell counts [ 20 – 22 , 27 ], and that quality-control cells containing different proportions of karyotypes can more accurately assess detection quality [ 26 ], we used 60 and 20% as the thresholds for aneuploidy detection [ 28 , 29 ]. We prepared high-, medium-, and low-value quality-control cells (A-H) with aneuploidy ratios of 10, 30, 70, and 90%, respectively, to provide a relatively consistent ratio of chimeras to the common clinical samples with various karyotypes as the basis to establish an ideal IQC for prenatal molecular diagnosis by FISH [ 20 – 24 ].…”

Genomic and structural abnormalities of the chromosome set of the fetus, depending on the age of pregnant women

“…Chromosomal preparations were developed according to the method of direct analysis from the chorion and placenta cones of the fetus, which were taken by the majority, after the umbilical cord blood lifocytes and amniocytes were grown in a nutrient medium under a thermostat for 72 hours, and the preparations were also colored according to GTG, CBG methods [12][13][14][15]. The study depends on the time of cultivation after obtaining the working material, the duration of preparation, and analysis.…”

Reappraisal of evolving methods in non-invasive prenatal screening: Discovery, biology and clinical utility

“…These techniques provided additional information with which to predict the overall genetic condition of the developing fetus [ [7] , [8] , [9] , [10] ]. Karyotyping and FISH have become the low-cost gold standard in prenatal screening of numerous chromosomal anomalies [ 11 , 12 ]. However, karyotyping and FISH exhibit certain limitations such as the need of a skilled analysts like cytogeneticists or clinical specialists who can obtain a tissue sample safely without harming the fetus.…”