Non-invasive prenatal tests for the detection of fetal aneuploidies are predominantly based on the analysis of cell-free DNA (cfDNA) from the plasma of pregnant women by next-generation sequencing. The development of alternative tests for routine genetic laboratories is therefore desirable. Multiplex digital droplet PCR was used to detect 16 amplicons from chromosome 21 and 16 amplicons from chromosome 18 as the reference. Two fluorescently labeled lock nucleic acid probes were used for the detection of reaction products. The required accuracy was achieved by examining 12 chips from each patient using Stilla technology. The plasma cfDNA of 26 pregnant women with euploid pregnancies and 16 plasma samples from pregnancies with trisomy 21 were analyzed to determine the cutoff value for sample classification. The test was validated in a blind study on 30 plasma samples from pregnant patients with a risk for trisomy 21 ranging from 1:4 to 1:801. The results were in complete agreement with the results of the invasive diagnostic procedure (sensitivity, specificity, PPV, and NPV of 100%). Low cost, and speed of analysis make it a potential screening method for implementation into the clinical workflow to support the combined biochemical and ultrasound results indicating a high risk for trisomy 21.

• MeSH
• aneuploidie MeSH
• Downův syndrom * diagnóza   genetika   MeSH
• lidé MeSH
• polymerázová řetězová reakce MeSH
• prenatální diagnóza metody   MeSH
• těhotenství MeSH
• trizomie MeSH
• volné cirkulující nukleové kyseliny * genetika   MeSH
• Check Tag
• lidé MeSH
• těhotenství MeSH
• ženské pohlaví MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• volné cirkulující nukleové kyseliny * MeSH

In families with X-linked recessive diseases, foetal sex is determined prenatally by detection of Y-chromosomal sequences in cell-free foetal DNA (cffDNA) in maternal plasma. The same procedure is used to confirm the cffDNA presence during non-invasive prenatal RhD incompatibility testing but there are no generally accepted markers for the detection of cffDNA fraction in female-foetus bearing pregnancies. We present a methodology allowing the detection of paternal X-chromosomal alleles on maternal background and the confirmation of female sex of the foetus by positive amplification signals. Using digital droplet PCR (ddPCR) we examined X-chromosomal INDEL (insertion/deletion) polymorphisms: rs2307932, rs16397, rs16637, rs3048996, rs16680 in buccal swabs of 50 females to obtain the population data. For all INDELs, we determined the limits of detection for each ddPCR assay. We examined the cffDNA from 63 pregnant women bearing Y-chromosome negative foetuses. The analysis with this set of INDELs led to informative results in 66.67% of examined female-foetus bearing pregnancies. Although the population data predicted higher informativity (74%) we provided the proof of principle of this methodology. We successfully applied this methodology in prenatal diagnostics in a family with Wiscott-Aldrich syndrome and in pregnancies tested for the risk of RhD incompatibility.

• MeSH
• analýza určování pohlaví metody   MeSH
• dospělí MeSH
• genetické testování MeSH
• lidé MeSH
• lidské chromozomy X genetika   MeSH
• mutace INDEL * MeSH
• plod chemie   metabolismus   MeSH
• polymerázová řetězová reakce metody   MeSH
• polymorfismus genetický * MeSH
• prenatální diagnóza metody   MeSH
• těhotenství MeSH
• volné cirkulující nukleové kyseliny analýza   genetika   MeSH
• Check Tag
• dospělí MeSH
• lidé MeSH
• těhotenství MeSH
• ženské pohlaví MeSH
• Publikační typ
• časopisecké články MeSH
• kazuistiky MeSH
• práce podpořená grantem MeSH
• Názvy látek
• volné cirkulující nukleové kyseliny MeSH

The cell-free DNA (cfDNA) is always present in plasma, and it is biomarker of growing interest in prenatal diagnostics as well as in oncology and transplantology for therapy efficiency monitoring. But does this cfDNA have a physiological role? Here we show that cfDNA presence and clearance in plasma of healthy individuals plays an indispensable role in immune system regulation. We exposed THP1 cells to healthy individuals' plasma with (NP) and without (TP) cfDNA. In cells treated with NP, we found elevated expression of genes whose products maintain immune system homeostasis. Exposure of cells to TP triggered an innate immune response (IIR), documented particularly by elevated expression of pro-inflammatory interleukin 8. The results of mass spectrometry showed a higher abundance of proteins associated with IIR activation due to the regulation of complement cascade in cells cultivated with TP. These expression profiles provide evidence that the presence of cfDNA and its clearance in plasma of healthy individuals regulate fundamental mechanisms of the inflammation process and tissue homeostasis. The detailed understanding how neutrophil extracellular traps and their naturally occurring degradation products affect the performance of immune system is of crucial interest for future medical applications.

• MeSH
• biologické markery krev   MeSH
• chromatografie kapalinová MeSH
• dospělí MeSH
• extracelulární pasti imunologie   MeSH
• hmotnostní spektrometrie MeSH
• krevní plazma MeSH
• lidé středního věku MeSH
• lidé MeSH
• mladý dospělý MeSH
• monocyty imunologie   MeSH
• přirozená imunita * MeSH
• sekvenční analýza hybridizací s uspořádaným souborem oligonukleotidů MeSH
• tandemová hmotnostní spektrometrie MeSH
• THP-1 buňky MeSH
• volné cirkulující nukleové kyseliny krev   MeSH
• zánět MeSH
• Check Tag
• dospělí MeSH
• lidé středního věku MeSH
• lidé MeSH
• mladý dospělý MeSH
• mužské pohlaví MeSH
• ženské pohlaví MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• biologické markery MeSH
• volné cirkulující nukleové kyseliny MeSH

Detection and characterization of circulating cell-free fetal DNA (cffDNA) from maternal circulation requires an extremely sensitive and precise method due to very low cffDNA concentration. In our study, droplet digital PCR (ddPCR) was implemented for fetal RHD genotyping from maternal plasma to compare this new quantification alternative with real-time PCR (qPCR) as a golden standard for quantitative analysis of cffDNA. In the first stage of study, a DNA quantification standard was used. Clinical samples, including 10 non-pregnant and 35 pregnant women, were analyzed as a next step. Both methods' performance parameters-standard curve linearity, detection limit and measurement precision-were evaluated. ddPCR in comparison with qPCR has demonstrated sufficient sensitivity for analysing of cffDNA and determination of fetal RhD status from maternal circulation, results of both methods strongly correlated. Despite the more demanding workflow, ddPCR was found to be slightly more precise technology, as evaluated using quantitative standard. Regarding the clinical samples, the precision of both methods equalized with decreasing concentrations of tested DNA samples. In case of cffDNA with very low concentrations, variance parameters of both techniques were comparable. Detected levels of fetal cfDNA in maternal plasma were slightly higher than expected and correlated significantly with gestational age as measured by both methods (ddPCR r = 0.459; qPCR r = 0.438).

• MeSH
• DNA krev   genetika   MeSH
• genotyp MeSH
• genotypizační techniky metody   MeSH
• gestační stáří MeSH
• krevní skupiny - systém Rh-Hr genetika   MeSH
• kvantitativní polymerázová řetězová reakce metody   MeSH
• lidé MeSH
• odběr vzorku krve metody   MeSH
• plod metabolismus   MeSH
• polymerázová řetězová reakce metody   MeSH
• prenatální diagnóza metody   MeSH
• reprodukovatelnost výsledků MeSH
• těhotenství MeSH
• Check Tag
• lidé MeSH
• těhotenství MeSH
• ženské pohlaví MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• srovnávací studie MeSH
• Názvy látek
• DNA MeSH
• krevní skupiny - systém Rh-Hr MeSH
• Rho(D) antigen MeSH Prohlížeč