Elena Ordoñez

Rapid prenatal diagnosis of common chromosome aneuploidies by QF‐PCR, results of 9 years of clinical experience

Despite being deliberately targeted to common chromosome aneuploidies, the rapid quantitative fluorescent polymerase chain reaction (QF‐PCR) tests can detect the majority of chromosome abnormalities in prenatal diagnosis. The main advantages of this assay are low cost, speed and automation allowing large‐scale application.

Concordance of Prevalence of Human Papillomavirus DNA in Anogenital and Oral Infections in a High-Risk Population

ABSTRACT The concordance of the prevalence of human papillomavirus (HPV) DNA in 188 sex workers in five different locations was investigated. HPV was found in 43.6% of the women, and its prevalence at genital sites was similar. Prevalence was highest among women aged 20 years or younger but declined thereafter in specimens from all anogenital sites.

Performance of screening for aneuploidies by cell‐free DNA analysis of maternal blood in twin pregnancies

To report clinical implementation of cell‐free DNA (cfDNA) analysis of maternal blood in screening for trisomies 21, 18 and 13 in twin pregnancies and examine variables that could influence the failure rate of the test.

Rapid prenatal diagnosis by QF-PCR: evaluation of 30,000 consecutive clinical samples and future applications.

Abstract:  Rapid prenatal diagnoses of major chromosome abnormalities can be performed on a large scale using highly polymorphic short tandem repeats (STRs) amplified by the quantitative fluorescent polymerase chain reaction (QF‐PCR). The assay was introduced as a preliminary investigation to remove the anxiety of the parents waiting for the results by conventional cytogenetic analysis using amniotic fluid or chorionic cells. However, recent studies, on the basis of the analyses of several thousand samples, have shown that this rapid approach has a very high rate of success and could reduce the need for cytogenetic investigations. Its high efficiency, for example, allows early interruption of affected fetuses without the need of waiting for completion of fetal karyotype. The main advantages of the QF‐PCR are its accuracy, speed, automation, and low cost that allows very large number of samples to be analyzed by few operators. Here, we report the results of using QF‐PCR in a large series of consecutive clinical cases and discuss the possibility that, in a near future, it may even replace conventional cytogenetic analyses on selected samples.

Clinical application of midtrimester non‐invasive fetal RHD genotyping and identification of RHD variants in a mixed‐ethnic population

This study aims to assess the suitability of non‐invasive prenatal RHD genotyping in non‐immunized midtrimester pregnant women from a mixed ethnic population, to prevent unnecessary anti‐D immunoglobulin prophylaxis and to identify RHD variants

Evaluation of Sample Stability and Automated DNA Extraction for Fetal Sex Determination Using Cell-Free Fetal DNA in Maternal Plasma

Objective. The detection of paternally inherited sequences in maternal plasma, such as the SRY gene for fetal sexing or RHD for fetal blood group genotyping, is becoming part of daily routine in diagnostic laboratories. Due to the low percentage of fetal DNA, it is crucial to ensure sample stability and the efficiency of DNA extraction. We evaluated blood stability at 4°C for at least 24 hours and automated DNA extraction, for fetal sex determination in maternal plasma. Methods. A total of 158 blood samples were collected, using EDTA-K tubes, from women in their 1st trimester of pregnancy. Samples were kept at 4°C for at least 24 hours before processing. An automated DNA extraction was evaluated, and its efficiency was compared with a standard manual procedure. The SRY marker was used to quantify cfDNA by real-time PCR. Results. Although lower cfDNA amounts were obtained by automated DNA extraction (mean 107,35 GE/mL versus 259,43 GE/mL), the SRY sequence was successfully detected in all 108 samples from pregnancies with male fetuses. Conclusion. We successfully evaluated the suitability of standard blood tubes for the collection of maternal blood and assessed samples to be suitable for analysis at least 24 hours later. This would allow shipping to a central reference laboratory almost from anywhere in Europe.

Screening for Sex Chromosome Aneuploidy by Cell-Free DNA Testing: Patient Choice and Performance

Objective: To study patient choice regarding testing for sex chromosome aneuploidy (SCA) and the performance of cell-free DNA (cfDNA) screening for SCA. Methods: Patient choice regarding screening for SCA and factors influencing this choice were evaluated in a single center. In a subsequent two-center study, cases that screened positive for SCA were analyzed to determine the positive predictive value (PPV) for each SCA. Results: In all, 1,957 (61.9%) of the 3,162 patients undergoing cfDNA testing opted for SCA screening. Regression analysis demonstrated that independent predictors of a patients decision for SCA were earlier gestational age, spontaneous conception, and cfDNA chosen as a primary method of screening. A total of 161 cases screened positive for SCA and follow-up data were available for 118 (73.3%). Forty-six of the 61 cases of 45,X were false-positive results and 15 were concordant with the fetal karyotype (PPV = 24.6%). Seventeen of the 22 cases of 47,XXX were false positive and 5 concordant (PPV = 22.7%). Eleven of the 30 cases of 47,XXY were false positive and 19 concordant (PPV = 63.3%). All 5 cases of 47,XYY were correctly identified, thus yielding a PPV of 100%. Conclusion: More than half of the patients undergoing cfDNA aneuploidy screening also opted for SCA testing, but they were less likely to do so in the presence of an increased risk of trisomy. SCAs involving the X chromosome had a lower PPV than those involving the Y chromosome.

Development and Validation of Multiplex Real-Time PCR Assay for Noninvasive Prenatal Assessment of Fetal RhD Status and Fetal Sex in Maternal Plasma

Objective: Noninvasive prenatal detection of RhD status and fetal sex is becoming part of daily practice in clinical laboratories. We evaluated a high throughput procedure for automated DNA extraction and developed a multiplex real-time PCR (rt-PCR) for the simultaneous detection of three fetal loci in a single reaction to assess fetal sex and RhD status in maternal plasma. Methods: An automated DNA extraction method was evaluated together with a new multiplex rt-PCR assay for the simultaneous detection of exons 5 and 7 of the RHD gene together with the Y chromosome marker DYS14 in maternal plasma. The test was evaluated on 60 samples of known fetal genotype obtained from RhD-negative pregnant women before being applied prospectively on 158 consecutive clinical cases. Results were compared with newborn phenotypes. Results: Automated DNA extraction allowed successful analysis of all samples. DYS14 was detected in 118 cases (male fetuses) and both RHD exon 5 and 7 were detected in 148 samples. In 70 samples neither RHD exon 5 nor RHD exon 7 were detected (RhD-negative fetuses). Absence of all three sequences (female RhD-negative fetuses) was assessed in 33 samples. All prenatal results were in concordance with postnatal RhD status and fetal sex without false- positive or -negative results. Conclusion: The automated DNA extraction procedure coupled with a novel multiplex rt-PCR assay proved accurate, efficient and reliable allowing rapid and high throughput noninvasive determination of fetal sex and RhD status in clinical samples.

Three-Year Follow-Up of a Prenatally Ascertained Apparently Non-Mosaic sSMC(10): Delineation of a Non-Critical Region

Small supernumerary marker chromosomes (sSMC) originating from chromosome 10 are rare and usually found in mosaic form. We present a de novo apparently non-mosaic sSMC(10) prenatally diagnosed in amniotic fluid and postnatally confirmed in peripheral blood. Characterization by array-CGH showed a pericentromeric duplication of 7.1 Mb of chromosome 10. The fetus did not show ultrasound abnormalities, and a normal female phenotype was observed during a 3-year postnatal follow-up. The absence of phenotypic abnormalities in the present case provides evidence of a non-critical pericentromeric region in 10p11.21q11.1 (hg19 35,355,570-42,448,569) associated with a duplication.