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. Key Points Hemolysis is not associated with lower proportions of fetal cell-free DNA in maternal blood samples drawn in cell-free DNA blood collection tubes. Laboratories performing prenatal cell-free DNA testing do not need to reject samples with visible hemolysis.

Performance of ImproGene Cell-Free DNA Tubes for Stabilization and Analysis of cfDNA in Blood Samples

“…noticeably increase when improper operation occurs and/or there is a longer preservation time. Some researchers have shown that the increased FHb concentration can reflect the destruction of red blood cells, which is not synchronized with the release of DNA from white blood cell lysis [23,24]. In our study, samples collected in ImproGene tubes showed the results consistent with the conclusion mentioned above while Streck tubes had only a weak correlation; the cfDNA concentration in EDTA tubes markedly increased with FHb concentration (Figure 1).…”

Lipid Metabolism Affects Fetal Fraction and Screen Failures in Non-invasive Prenatal Testing

“…Second, other researchers have studied the effects of maternal and fetal characteristics and experimental factors on fetal fraction with the aim of adjusting these factors to obtain higher numbers of fetal-derived cfDNA. Fetal fraction can be influenced by maternal body mass index (BMI), gestational age (GA), anticoagulation therapy, blood collection, and fetal aneuploidy ( 14 , 18 , 19 ). Based on prior research, one potential way to reduce the incidence of screening failure is to repeat test failures by sequencing a second sample.…”

The effect of hemolysis on quality control metrics for noninvasive prenatal testing

“…The influence of hemolysis on NIPT has not been well investigated. A recent study showed that hemolysis in Cell-Free DNA Collection Tubes had no impact on the fetal fraction in NIPT [ 19 ]. One of the limitations of this study was that the hemoglobin concentration was assessed visually rather than with automated spectrophotometric instrumentation.…”