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

ABSTRACT: 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.

SUBMITTER: Stokowski R

PROVIDER: S-EPMC7113221 | biostudies-literature | 2020 Apr

REPOSITORIES: biostudies-literature

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

Stokowski Renee R White Karen K Hacker Coleen C Doshi Jigna J Schmid Maximilian M

Molecular diagnosis & therapy 20200401 2

<h4>Background</h4>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.<h4>Objective</h4>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.<h4>Methods</h4>In the first part of the study, ...[more]

PMID: 32056107

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Project description:While circulating cell-free DNA (cfDNA) is becoming a powerful marker for noninvasive identification of infectious pathogens in liquid biopsy specimens, a microbial cfDNA baseline in healthy individuals is urgently needed for the proper interpretation of microbial cfDNA sequencing results in clinical metagenomics. Because noninvasive prenatal testing (NIPT) shares many similarities with the sequencing protocol of metagenomics, we utilized the standard low-pass whole-genome-sequencing-based NIPT to establish a microbial cfDNA baseline in healthy people. Sequencing data from a total of 107,763 peripheral blood samples of healthy pregnant women undergoing NIPT screening were retrospectively collected and reanalyzed for microbiome DNA screening. It was found that more than 95% of exogenous cfDNA was from bacteria, 3% from eukaryotes, and 0.4% from viruses, indicating the gut/environment origins of many microorganisms. Overall and regional abundance patterns were well illustrated, with huge regional diversity and complexity, and unique interspecies and symbiotic relationships were observed for TORCH organisms (Toxoplasma gondii, others [Treponema pallidum {causing syphilis}, hepatitis B virus {HBV}, and human parvovirus B19 {HPV-B19}], rubella virus, cytomegalovirus [CMV], and herpes simplex virus [HSV]) and another common virus, Epstein-Barr virus (EBV). To sum up, our study revealed the complexity of the baseline circulating microbial cfDNA and showed that microbial cfDNA sequencing results need to be interpreted in a more comprehensive manner. IMPORTANCE While circulating cell-free DNA (cfDNA) has been becoming a powerful marker for noninvasive identification of infectious pathogens in liquid biopsy specimens, a baseline for microbial cfDNA in healthy individuals is urgently needed for the proper interpretation of microbial cfDNA sequencing results in clinical metagenomics. Standard low-pass whole-genome-sequencing-based NIPT shares many similarities with the sequencing protocol for metagenomics and could provide a microbial cfDNA baseline in healthy people; thus, a reference cfDNA data set of the human microbiome was established with sequencing data from a total of 107,763 peripheral blood samples of healthy pregnant women undergoing NIPT screening. Our study revealed the complexity of circulating microbial cfDNA and indicated that microbial cfDNA sequencing results need to be interpreted in a more comprehensive manner, especially with regard to geographic patterns and coexistence networks.

Project description:ObjectiveTo assess the detection efficiency of noninvasive prenatal testing (NIPT) for fetal autosomal aneuploidy, sex chromosome aneuploidy (SCA), other chromosome aneuploidy, copy number variation (CNV), and to provide further data for clinical application of NIPT.Materials and methods25,517 pregnant women who underwent NIPT testing in Anhui Province Maternity and Child Health Hospital from September 2019 to September 2020 were selected, and samples with high-risk test results were subjected to karyotype analysis for comparison by using amniotic fluid, with some samples subjected to further validation by chromosomal microarray analysis, and followed up for pregnancy outcome.ResultsA total of 25,517 pregnant women who received NIPT, 25,502 cases were tested successfully, and 294 high-risk samples (1.15%) were detected, there were 96 true positive samples, 117 false positive samples and 81 cases were refused further diagnosis. Samples with high risk of autosomal aneuploidy were detected in 71 cases (0.28%), and 51 cases were confirmed, including: trisomy 21 (T21) in 44 cases, trisomy 18 (T18) in 5 cases, and trisomy 13 (T13) in 2 cases; the positive predictive value (PPV) was 91.67%, 45.45%, and 33.33%, respectively, and the negative predictive value was 100%, the false positive rate (FPR) was 0.02%, 0.02%, and 0.02%, respectively.13 samples with high risk of mosaic trisomies 21, 18, and 13 were detected, and 1 case of T21mos was confirmed with a PPV of 8.33%. Samples with high risk of SCA were detected in 72 cases (0.28%), and the diagnosis was confirmed in 23 cases, with a PPV of 41.07% and a FPR of 0.13%. These included 3 cases of 45,X, 6 cases of 47,XXY, 8 cases of 47,XXX and 6 cases of 47,XYY, with PPVs of 12.00%, 50.00%, 72.73%, and 75.00%, respectively, and false-positive rates of 0.09%, 0.02%, 0.01% and 0.01% respectively. Samples with high risk of CNV were detected in 104 cases (0.41%) and confirmed in 18 cases, with a PPV of 32.14% and a FPR of 0.15%. Samples with high risk of other chromosomal aneuploidy were detected in 34 cases (0.13%), and the diagnosis was confirmed in 3 cases, which were T2, T9, and T16 respectively. The overall PPV for other chromosome aneuploidy was 12.50%, with a FPR of 0.08%.ConclusionNIPT is indicated for trisomies 21, 18 and 13 screening, especially for T21. It also has some certain reference value for SCA and CNV, but is not recommended for screening of other chromosomal aneuploidy.

Dataset Information

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

Publications

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

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