Impact of long‐term storage of plasma and cell‐free DNA on measured DNA quantity and fetal fraction

Clausen, Frederik Banch; Barrett, Angela N.; Advani, Henna V.; Choolani, Mahesh; Dziegiel, Morten Hanefeld

doi:10.1111/vox.12923

Cited by 8 publications

(6 citation statements)

References 32 publications

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“…Owing to maternal and fetal DNA fluctuations, FF in maternal plasma showed a downward trend after 2 years of storage at −25 • C, decreasing after 3 years. However, the FF of the extracted DNA stored at −25 • C for 18 months did not change, and the FF of the extracted DNA stored for 3 years increased by 27% compared with that detected immediately (83). After freezing, the plasma GC content in samples from pregnant women increased, and the FF, unit reads/total reads ratio, and Z score of trisomy 21 in male fetuses decreased, resulting in decreased detection accuracy (84).…”

Section: Experimental Factorsmentioning

confidence: 94%

Factors Affecting the Fetal Fraction in Noninvasive Prenatal Screening: A Review

Deng

Liu

2022

Front. Pediatr.

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A paradigm shift in noninvasive prenatal screening has been made with the discovery of cell-free fetal DNA in maternal plasma. Noninvasive prenatal screening is primarily used to screen for fetal aneuploidies, and has been used globally. Fetal fraction, an important parameter in the analysis of noninvasive prenatal screening results, is the proportion of fetal cell-free DNA present in the total maternal plasma cell-free DNA. It combines biological factors and bioinformatics algorithms to interpret noninvasive prenatal screening results and is an integral part of quality control. Maternal and fetal factors may influence fetal fraction. To date, there is no broad consensus on the factors that affect fetal fraction. There are many different approaches to evaluate this parameter, each with its advantages and disadvantages. Different fetal fraction calculation methods may be used in different testing platforms or laboratories. This review includes numerous publications that focused on the understanding of the significance, influencing factors, and interpretation of fetal fraction to provide a deeper understanding of this parameter.

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Section: Experimental Factorsmentioning

confidence: 94%

Factors Affecting the Fetal Fraction in Noninvasive Prenatal Screening: A Review

Deng

Liu

2022

Front. Pediatr.

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“…Given that these samples were collected from the same dogs (paired samples from the same tube were stored either on ice or at room temperature) and that they had been centrifuged in the same tube before these paired aliquots were prepared, it is not possible for the room temperature samples to truly have higher nucleosome concentrations than samples stored on ice, rather it more likely that there is a minor temperature associated conformational change that enables better access of the antibody to the nucleosome. These differences were not statistically significant and this finding is not specific to our study as increased levels of DNA have been seen in biobank samples after long term storage as a result of protein disassociation allowing more DNA to be available for PCR amplification [28]. Thus, to ensure the most accurate results are generated it is recommended that samples be shipped on ice as these concentrations were most similar to time 0 nucleosome concentrations in other assays.…”

Section: Plos Onementioning

confidence: 71%

Evaluation of nucleosome concentrations in healthy dogs and dogs with cancer

et al. 2020

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Introduction Nucleosomes consist of small fragments of DNA wrapped around a histone octamer core. Diseases such as cancer or inflammation lead to cell death, which causes fragmentation and release of nucleosomes into the blood. The Nu.Q™ technology measures circulating nucleosome levels and exploits the different compositions of cancer derived nucleosomes in blood to detect and identify cancer even at early stages. The objectives of this study are to identify the optimal sample type for the Nu.Q™ H3.1 assay and to determine if it can accurately detect nucleosomes in the blood of healthy canines as well as those with cancer. Materials and methods Blood samples from healthy canine volunteers as well as dogs newly diagnosed with lymphoma were used. The blood was processed at a variety of times under a variety of conditions to determine the most reliable sample type and conditions, and to develop an appropriate processing strategy to ensure reliably accurate results. Results Nucleosomes could be detected using a variety of sample collection and processing protocols. Nucleosome signals were highest in EDTA plasma and serum samples and most consistent in plasma. Samples should be processed within an hour of collection. Experiments showed that samples were able to withstand several freeze thaw cycles. Processing time and tcollection tube type did affect nucleosome detection levels. Finally, significantly elevated concentrations of nucleosomes were seen in a small cohort of dogs that had been newly diagnosed with lymphoma. Conclusions When samples are collected and processed appropriately, the Nu.Q™ platform can reliably detect nucleosomes in the plasma of dogs. Further testing is underway to validate and optimize the Nu.Q™ platform for veterinary use.

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“…The blood tubes, were in most cases, put directly onto the QIAsymphony (QIAGEN, Hilden, Germany) instrument for DNA extraction but for samples close to 5 days before they could be analysed, plasma was transferred to cryotubes and frozen at −20°C until further analysis. As shown by others, the use of short‐time frozen plasma does not have a major effect on results [ 13 ].…”

Section: Methodsmentioning

confidence: 95%

Single‐exon approach to non‐invasive fetal RHD screening in early pregnancy: An update after 10 years' experience

et al. 2022

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Background and Objectives Anti‐D prophylaxis, administered to RhD‐negative women, has significantly reduced the incidence of RhD immunization. Non‐invasive fetal RHD screening has been used in Stockholm for more than 10 years to identify women who will benefit from prophylaxis. The method is based on a single‐exon approach and is used in early pregnancy. The aim of this study was to update the performance of the method. Materials and Methods The single exon assay from Devyser AB is a multiplex kit detecting both exon 4 of the RHD gene and the housekeeping gene GAPDH. Cell‐free DNA was extracted from 1 ml of plasma from EDTA blood taken during early pregnancy, weeks 10–12. The genetic RHD results were compared with serological typing of newborns for a determination of sensitivity and specificity. Results In total, 4337 pregnancies were included in the study; 44 samples (1%) were inconclusive either due to maternal RHD gene variants (n = 34) or technical reasons (n = 10). Of the remaining 4293 pregnancies, a total number of nine discrepant results were found. False positive results (n = 7) were mainly (n = 4) due to RHD gene variants in the child. False‐negative results were found in two cases, of which one was caused by a technical error. None of the false‐negative cases was due to RHD gene variants. Overall, the sensitivity of the method was 99.93% and specificity 99.56%. Conclusion The single‐exon assay used in this study is correlated with high sensitivity and specificity.

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Impact of long‐term storage of plasma and cell‐free DNA on measured DNA quantity and fetal fraction

Cited by 8 publications

References 32 publications

Factors Affecting the Fetal Fraction in Noninvasive Prenatal Screening: A Review

Factors Affecting the Fetal Fraction in Noninvasive Prenatal Screening: A Review

Evaluation of nucleosome concentrations in healthy dogs and dogs with cancer

Single‐exon approach to non‐invasive fetal RHD screening in early pregnancy: An update after 10 years' experience

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