Effects of Pre-analytical Variables on Cell-free DNA Extraction for Liquid Biopsy

BACKGROUND: Extraction of cell-free DNA (cfDNA) is a key step for determining the quality of cfDNA-related molecular diagnostics. We evaluated the effect of sample containers and sample storage conditions on cfDNA extraction. METHODS: The cfDNA extraction using the MagMAX Cell-Free DNA Isolation Kit from five healthy controls and five lung cancer patients was evaluated according to the type of sample container and storage conditions: K2-EDTA container, <1, 6, 24, and 48 hr storage at 4℃ after immediate plasma separation; and Cell-Free DNA BCT container, <1, 3, 7, and 14 days stored at room temperature. Mutation analysis of EGFR exons 18–21 was performed. To assess the effect of a delay in centrifugation, EDTA whole blood samples from five healthy individuals were stored at 4℃ for 6, 12, and 24 hr before plasma separation. RESULTS: There was no significant difference in the amount and nucleic acid size of cfDNA in both controls and patients with cancer when EDTA plasma was stored at 4℃ up to 48 hr. The amount and size of cfDNA in the BCT container were not different up to 7 days; however, the 14-day sample showed an increase in cfDNA concentration due to genomic DNA contamination. EGFR mutations were detected on EDTA containers up to 48 hr and with BCT containers up to 14 days. When EDTA whole blood was stored at 4℃ and plasma separation was delayed, the cfDNA concentration increased from 24 hr. CONCLUSIONS: The cfDNA extraction was affected by the sample containers and storage conditions.

Contamination of pulse oximeter probes before and after decontamination in two intensive care units

Background. The internal surfaces of pulse oximeter probes may be overlooked as hot spots for pathogenic microorganisms in an intensive care unit (ICU), thereby contributing to the high incidence of hospital-acquired infections. Objectives. To determine the growth and identification of microorganisms on pulse oximeter probes in the multidisciplinary ICU (MICU) at Charlotte Maxeke Johannesburg Academic Hospital and the burns ICU (BICU) at Chris Hani Baragwanath Academic Hospital, before and after decontamination. Methods. This was a cross-sectional, comparative and contextual study, using purposive sampling. Data were collected from the internal surfaces of 34 pulse oximeter probes in a MICU and BICU. Each pulse oximeter probe was swabbed before and after decontamination. The endemic microorganism profile for the two ICUs was obtained from a laboratory database. Results. Internal surfaces of 31 (91%; 95% confidence interval (CI) 0.76 - 0.98) pulse oximeter probes were contaminated with 9 different pathogenic microorganisms pre decontamination. Acinetobacter baumannii, Klebsiella pneumoniae and Pseudomonas aeruginosa were endemic to both ICUs, and were the most-frequently isolated microorganisms. Staphylococcus aureus was the most common microorganism endemic to both ICUs, isolated on the internal surfaces of only 2 pulse oximeter probes. Of the internal surfaces of pulse oximeter probes, 6 (18%; 95% CI 0.07 - 0.35) remained contaminated post decontamination, with a microorganism growth reduction of 80% (p=0.0001). Conclusion. The internal surfaces of pulse oximeter probes may serve as hot spots for an array of pathogens with the potential to cause infection and outbreaks in ICUs. Decontamination of the internal surfaces of pulse oximeter probes should be emphasised

Application of denaturing high performance liquid chromatography for the detection of maternal DNA contamination during prenatal diagnosis / 中华医学遗传学杂志

<p><b>OBJECTIVE</b>To establish a high-quality method for detecting short tandem repeats(STR) using denaturing high performance liquid chromatography(DHPLC) in order to exclude maternal contamination and improve the accuracy of prenatal diagnosis.</p><p><b>METHODS</b>Two families were recruited. DNA was extracted from blood samples from the parents as well as amniotic fluid. Sixteen STR sites were amplified and analyzed based on the range of allele length reported by a STR database. Maternal DNA was mixed with DNA derived from amniotic fluid samples with the ratio 1:1, 1:4, 1:9, 1:19 and 1:99. vWA STR site was detected with DHPLC to confirm the sensitivity of detection.</p><p><b>RESULTS</b>Sixteen STR sites were analyzed by DHPLC, for which at least 10 were found to be different between the mothers and fetuses. The detection rate, with maternal contamination excluded, was 66.7%. And the sensitivity of detection was 1-10%.</p><p><b>CONCLUSION</b>Maternal contamination of amniotic fluid can be rapidly excluded with accuracy with DHPLC, which features a high sensitivity and good quality control, and can meet the European standards and provide a reliable quality control platform for prenatal diagnosis.</p>

Retal Sex Determination by Polymerase Chain Reaction / 대한산부인과학회잡지

Ror fetal sex determination by the PCR method, oilgoprimers to Y- chromosome gene, DYZI, SRY, and AMGL were synthesized genomic DNA was extracted from male and female placenta for the control use. DYZI represented 154 bp single band to 0.001 pg/ml male genomic DNA but did not represent 154 bp band in female genomic DNA, SRY represented 341 bp bandto 1 pg/ml male genomic DNA in 2% agarose gel eleftrophoresis stained with ethidium bromide. DYZI was 1,000 fold sensitive than Sry and AMGL. DYZI and SRY could not identify the PDR failure from female but AMGL identified to 1,000-fold. During the dyal ampiification of female genomic DNA mixed with male genomic DNA, 0.00125 pg/nl, 1:400 part, male genomic DNA contamination represented male band but SRY amplification did not represent male band. It was suggested that SRY gene was deleted in two 46,XY felmle cases. For fetal sex determination, PCR with DYZL, SRY, and AMGL was performed in 10 cases. For fetal sex determination, PCR with DYZL, SRY, and AMGL with karyotyping in 10 cases of chorionic villi sex dietermination, PCR with DYZI, SRY, and AMGL was performed in 10 cases. For feral sex determination, PCR with DYZI, SRY, and AMGL with karyothping result, fetal sex determination, PCR with DYZI, SRY, and AMGL was performed in 10 Cases of choricinic villi and 15 cases of amnionic cells. By the comparison with karyotyping result, fetal sex determination was achieved successfully in all 23 samlies using PCR of SRY and AMGL but false result was detected in 3 cases(13%) using DYZI. Acording to our results, it was concluded that DYZL was 1,000-fold sensitive than SRY and AMGL but could not be used because of its false results, and AMGL and SRY must be used concomitantly for precise sex determination.