A microfluidic platform for high-purity cell free DNA extraction from plasma for non-invasive prenatal testing.

OBJECTIVES: Increase the yield and purity of cell-free DNA (cfDNA) extracted from plasma for non-invasive prenatal testing (NIPT) as inefficiencies in this extraction and purification can dramatically affect the sensitivity and specificity of the test. METHODS: This work integrates cfDNA extraction from plasma with a microfluidic chip platform by combining magnetic bead-based extraction and electroosmotic flow on the microfluidic chip. Various wash buffers and voltage conditions were simulated using COMSOL Multiphysics Modeling and tested experimentally. RESULTS: When performing the first wash step of this assay on the microfluidic chip with 300 V applied across the channel there was a six-fold increase in the A260 /A230 ratio showing a significant improvement (p value 0.0005) in the purity of the extracted sample all while maintaining a yield of 68.19%. These values are critical as a high yield results in more sample to analyze and an increase in A260 /A230 ratio corresponds to a decrease in salt contaminants such as guanidinium thiocyanate which can interfere with downstream processes during DNA library preparation and potentially hinder the NIPT screening results. CONCLUSIONS: This technique has the potential to improve NIPT outcomes and other clinically relevant workflows that use cfDNA as an analyte such as cancer detection.

Noninvasive prenatal testing: Advancing through a virtuous circle of science, technology and clinical applications.

BACKGROUND: Since the discovery of cell-free fetal DNA in maternal blood in 1997, the interplay of basic scientific observations and technological developments have continued to drive new clinical applications in the field. AIMS: This commentary discusses a number of examples in this virtuous circle of science, technology and clinical applications. MATERIALS & METHODS: Commentary and literature review. RESULTS: One example of technological developments is the detection technologies for detecting circulating DNA, moving from conventional PCR, to real-time PCR, to massively parallel sequencing. One example of basic scientific understanding is the size and fragmentation patterns of circulating DNA. DISCUSSION: Beyond creating a global paradigm in prenatal medicine, the development of noninvasive prenatal testing has also impacted other areas such as cancer screening and transplantation monitoring. Finally, the commentary looks forward to what might be in store in the next decade.

Non-invasive prenatal testing suggesting a maternal malignancy: What do we tell the prospective parents in Belgium?

Cancer is diagnosed in one in 1000 to 1500 pregnancies. Most frequently encountered malignancies during pregnancy are breast cancer, hematological cancer, cervical cancer and malignant melanoma. Maternal cancer is associated with an increased risk of IUGR and preterm labor, especially in patients with systemic disease or those receiving chemotherapy during pregnancy, requiring a high-risk obstetrical follow-up. Fetal aneuploidy screening by non-invasive prenatal testing (NIPT) can lead to the incidental identification of copy number alterations derived from non-fetal cell-free DNA (cfDNA), as seen in certain cases of maternal malignancy. The identification of tumor-derived cfDNA requires further clinical, biochemical, radiographic and histological investigations to confirm the diagnosis. In such cases, reliable risk estimation for fetal trisomy 21, 18 and 13 is impossible. Therefore, invasive testing should be offered when ultrasonographic screening reveals an increased risk for chromosomal anomalies, or when a more accurate test is desired. When the fetal karyotype is normal, long term implications for the fetus refer to the consequences of the maternal disease and treatment during pregnancy. This manuscript addresses parental questions when NIPT suggests a maternal malignancy. Based on current evidence and our own experience, a clinical management scheme in a multidisciplinary setting is proposed.

The role of fetal echocardiogram after detection of extracardiac anomalies in utero (fetal echocardiogram for extracardiac malformations).

OBJECTIVE: We aimed to investigate the utility of comprehensive screening fetal echocardiography (FE) for patients diagnosed with any type of fetal extracardiac malformation (ECM) at a single multidisciplinary fetal center. METHODS: We retrospectively reviewed all patients presenting to our referral center for FE due to a prenatal diagnosis of ECM (January 2013-December 2018). RESULTS: Among 641 patients with ≥1 ECM referred for FE, 78 (12.2%) had CHD diagnosed at 25.6 ± 0.5 weeks. The frequency of CHD by type of ECM ranged from 35.1% for craniofacial to 9.8% for thoracic. Increasing number of fetal ECMs was strongly associated with CHD: odds ratio 2.01 (95% confidence interval: 1.06-3.69) for two ECMs, 9.57 (2.00-49.05) for three ECMs, and 11.68 (3.84-37.15) for more than three ECMs. Of fetuses with ECM and an abnormal genetic finding, 33.3% had CHD as compared to 10.9% of those without (p < 0.0001). Obstetric anatomy sonogram detected 43.6% of CHD. CONCLUSION: CHD was commonly diagnosed among fetuses with any type of ECM at our center but was not always detected on obstetric sonogram. As the presence of CHD may impact decision-making and perinatal care, patients with a diagnosis of any fetal ECM should be considered for FE.

Non-invasive prenatal testing in the management of twin pregnancies.

Twin pregnancies are common and associated with pregnancy complications and adverse outcomes. Prenatal clinical management is intensive and has been hampered by inferior screening and less acceptable invasive testing. For aneuploidy screening, meta-analyses show that non-invasive prenatal testing (NIPT) through analysis of cell-free DNA (cf-DNA) is superior to serum and ultrasound-based tests. The positive predictive value for NIPT is driven strongly by the discriminatory power of the assay and only secondarily by the prior risk. Uncertainties in a priori risks for aneuploidies in twin pregnancies are therefore of lesser importance with NIPT. Additional information on zygosity can be obtained using NIPT. Establishing zygosity can be helpful when chorionicity was not reliably established early in pregnancy or where the there is a concern for one versus two affected fetuses. In dizygotic twin pregnancies, individual fetal fractions can be measured to ensure that both values are satisfactory. Vanishing twins can be identified by NIPT. Although clinical utility of routinely detecting vanishing twins has not yet been demonstrated, there are individual cases where cf-DNA analysis could be helpful in explaining unusual clinical or laboratory observations. We conclude that cf-DNA analysis and ultrasound have synergistic roles in the management of multiple gestational pregnancies.

Overview and recent developments in cell-based noninvasive prenatal testing.

Investigators have long been interested in the natural phenomenon of fetal and placental cell trafficking into the maternal circulation. The scarcity of these circulating cells makes their detection and isolation technically challenging. However, as a DNA source of fetal origin not mixed with maternal DNA, they have the potential of considerable benefit over circulating cell-free DNA-based noninvasive prenatal genetic testing (NIPT). Endocervical trophoblasts, which are less rare but more challenging to recover are also being investigated as an approach for cell-based NIPT. We review published studies from around the world describing both forms of cell-based NIPT and highlight the different approaches' advantages and drawbacks. We also offer guidance for developing a sound cell-based NIPT protocol.

Stimuli-Responsive Microfluidic Interface Enables Highly Efficient Capture and Release of Circulating Fetal Cells for Non-Invasive Prenatal Testing.

Circulating fetal nucleated cells (CFCs) carrying whole genomic coding of the fetus in maternal blood have been pursued as ideal biomarkers for noninvasive prenatal testing (NIPT). However, a significant limitation is the need to enrich sufficient cells in quantity and purity for fetal genetic disorder diagnosis. This study for the first time demonstrates a stimuli-responsive ligand enabling interface on array patterned microfluidic chip (NIPT-Chip) for high efficient isolation and release of CFCs in untreated whole blood. Deterministic lateral displacement (DLD)-array was patterned in the chip to increase collision frequency between CFCs and surface-anchored antibody to achieve high efficient cell capture. More importantly, the stimuli-responsive interface enables gentle release of captured CFCs through a thiol exchange reaction for downstream gene analysis of NIPT. With the advantages of simple processing, efficient isolation, and gentle release, NIPT-Chip offers great potential for clinical translation of circulating fetal cell-based NIPT.

Hemolysis and Fetal Fraction in Cell-Free DNA Blood Collection Tubes for Noninvasive Prenatal Testing.

BACKGROUND: Lysis of maternal white blood cells in prenatal cell-free DNA (cfDNA) test samples increases the level of maternal DNA and consequently decreases fetal fraction. OBJECTIVE: The objective of this study was to determine whether hemolysis, traditionally used as a marker for cell lysis, is correlated with a decrease in fetal fraction in maternal blood samples collected in specialized cfDNA tubes for noninvasive prenatal testing. METHODS: In the first part of the study, blood from pregnant women was collected into three Roche Cell-Free DNA Collection Tubes. These replicate specimens from the same subject were evaluated for a visual difference in hemoglobin level as a measure of hemolysis. The specimens were then processed with the Harmony® prenatal test to measure fetal fraction using polymorphic digital analysis of selected regions (DANSR) assays. In a second part of the study, clinical laboratory samples with hemoglobin levels of ≥ 500 mg/dL were tracked through the laboratory and their fetal fraction compared with that of concurrently processed samples with lower hemoglobin levels. RESULTS: There was no significant difference in fetal fraction in 339 paired samples, with a difference in hemoglobin levels ranging from 0 to 1000 mg/dL. There was strong correlation in fetal fraction between tubes, regardless of the differences in hemoglobin concentration. The fetal fraction distribution in 203 tracked clinical samples with hemoglobin levels ≥ 500 mg/dL was statistically equivalent to the distribution in a concurrent series of 12,705 samples. CONCLUSION: Hemolysis in maternal blood samples collected in specialized cfDNA tubes does not correlate with a decrease in fetal fraction; therefore, it should not be a cause for rejection of samples submitted for prenatal cfDNA testing.