Integrated Workflow for Blood Collection, ccfDNA Stabilization … · 2020-01-05 · Integrated...
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Integrated Workflow for Blood Collection, ccfDNA Stabilization and Extraction for Non-Invasive Prenatal Testing 1 Andrea Ullius, 1 Thorsten Voss, 2 Stephan Busche, 2 Wera Hofmann, 1 Daniel Grölz 1 PreAnalytiX GmbH, Hilden, Germany; 2 LifeCodexx, Konstanz, Germany Background Non-invasive prenatal testing (NIPT) uses fetal circulating cell-free DNA (ccfDNA) in the maternal blood circulation. The release of maternal genomic DNA from blood cells during blood collection, transport and storage significantly reduces the sensitivity of the next-generation sequencing (NGS) based NIPT assays. Moreover, the reliable and reproducible extraction of the low abundance and short fetal ccfDNA fragments is technically challenging. Both difficulties are addressed by the PAXgene ® Blood ccfDNA System*, which consists of a blood collection tube (PAXgene Blood ccfDNA Tube*) and a ccfDNA purification kit (QIAsymphony ® PAXgene Blood ccfDNA Kit*). The PAXgene Blood ccfDNA Tube uses a plastic BD Vacutainer ® tube with a unique, non-crosslinking chemistry for stabilization of blood cells. The QIAsymphony PAXgene Blood ccfDNA Kit can be used for automated isolation of either small ccfDNA fragments or co-isolation of small and large ccfDNA fragments. The performance of the new system was evaluated in two research studies in comparison to K 2 EDTA tubes and the Streck Cell-Free DNA BCT ® . PAXgene Blood ccfDNA Tube (RUO) Tube features: • Effective stabilization of: – White blood cells – Helps prevent release of gDNA – Red blood cells – Helps minimize hemolysis • Helps maximize ccfDNA yield from plasma, minimize background gDNA • Non-crosslinking, no DNA modification • BD Vacutainer plastic tube with BD Hemogard™ safety closure – Helps minimize risk of tube breakage – Enhances safety for healthcare and lab personnel – Helps minimize contamination between samples – Consistent blood draw volume QIAsymphony PAXgene Blood ccfDNA Kit (RUO) Kit features: • Dedicated ccfDNA isolation technology for use with the PAXgene Blood ccfDNA Tube – Binding chemistry optimized for use with PAXgene Blood ccfDNA Tube reagent – Optimized input volumes to accommodate higher volume plasma • Two protocol lines – Standard protocol for preferred extraction of small fragments (≤ 500 bp) with 60 µl elution volume – Large fragment protocol for co-isolation of large fragments (> 500 bp) with flexible elution volume (60, 100, 150 µl) • 2.4 ml or 4.8 ml plasma processing • Prefilled cartridges are ready to use QIAsymphony PAXgene Blood ccfDNA Protocols Standard Protocol Line (STA) (small fragments preferred) 2.4 or 4.8 ml plasma protocol Large Fragment Protocol Line (LAF) (efficient co-isolation of large fragments) 2.4 or 4.8 ml plasma protocol Binding/proteinase K digestion with Buffer BCD1 Binding/proteinase K digestion with Buffer BCD2 Wash 1 Wash 2 Wash 3 Elution 60 μl Elution 60/100/150 μl Wash 1 Wash 2 Wash 3 Whole blood collected, transported and stored in PAXgene Blood ccfDNA Tubes Plasma generation Automated ccfDNA isolation Figure 1. Standard and large fragment protocol lines. With the standard protocols (STA) predominately small ccfDNA fragments are isolated while large fragment protocols (LAF) efficiently co-purify large and small ccfDNA fragments. Both protocol lines consist of protocols for 2.4 ml or 4.8 ml plasma input. Disclaimer *For Research Use Only. Not for use in diagnostic procedures. For up-to-date licensing information and product-specific disclaimers, see the respective PreAnalytiX or QIAGEN handbook or user manual. PreAnalytiX or QIAGEN kit handbooks and user manuals are available at www.preanalytix.com or www.qiagen.com or can be requested from QIAGEN Technical Services or your local distributor. Trademarks: QIAGEN ® , QIAamp ® , QIAsymphony ® (QIAGEN Group); PAXgene ® (PreAnalytiX GmbH), BD Hemogard™, BD Vacutainer ® (Becton, Dickinson and Company), PrenaTest ® , QuantYfeX ® (LifeCodexx AG); HiSeq™ (Illumina, Inc.); Cell-Free DNA BCT ® (Streck, Inc.). Registered names, trademarks, etc. used in this document, even when not specifically marked as such, are not to be considered unprotected by law. © 2016 PreAnalytiX GmbH, all rights reserved. Study 2: Validation of the PAXgene Blood ccfDNA System for non-invasive prenatal testing (NIPT) application Study Setup: • Blood samples were collected from 62 consented pregnant women • Two blood collection tubes were collected per patient: – Tube 1: Streck Cell-Free DNA BCT – Tube 2: PAXgene Blood ccfDNA Tube • Plasma processing from half of the blood samples collected into PAXgene tubes was performed with one centrifugation step, the remainder with two centrifugation steps • All samples underwent the regular PrenaTest procedure per the standard assay protocol • 25 plasma samples collected into PAXgene Blood ccfDNA Tubes were selected for downstream analysis (12 with one, 13 with two centrifugation steps) 0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 Fetal ccfDNA (%) Sample Number QuantYfeX: Relative Fetal ccfDNA Content Streck BCT PAXgene Figure 5: Relative fetal fraction of paired plasma samples collected in PAXgene Blood ccfDNA Tubes or Streck Cell-Free DNA BCT tubes. Relative amount of fetal ccfDNA in maternal background was quantified with methylation sensitive QuantYfeX QC Assay. Relative fetal ccfDNA fractions (PAXgene median: 13.7%; Streck BCT median: 12.4%) in maternal blood were not significantly different between the two different tubes (p-value: 0.20 paired T-Test, two-tailed distribution). 86 87 88 89 90 91 92 93 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 Mapped Reads (%) Sample Number NGS: Mapped Reads [%] A. B. Streck BCT PAXgene 80 81 82 83 84 85 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 Unique Mapped Reads (%) Sample Number NGS: Unique Mapped Reads [%] Streck BCT PAXgene Figure 6: NGS read out quality for paired plasma samples collected in PAXgene Blood ccfDNA Tubes or Streck Cell-Free DNA BCT tubes. ccfDNA from Streck BCTs and PAXgene tubes sequenced on Illumina HiSeq™ with NEB library preparation. There were significantly more mapped reads with ccfDNA from PAXgene tubes (p-value: 1.78E-04, two-tailed paired T-test; A). There was no significant difference in unique mapped reads (B). Study 2 Results 1. Relative fetal ccfDNA fractions in maternal blood in both tubes were not significantly different 2. Significantly more mapped reads with ccfDNA from PAXgene tubes 3. No significant difference in uniquely mapped reads 4. No significant difference with PAXgene tube samples processed with one or two centrifugation steps 5. All samples which passed the internal quality standards produced equivalent results with the PrenaTest assay Conclusions Study 1: • PAXgene Blood ccfDNA Tubes stabilize whole blood samples for up to 7 days of storage at room temperature (15–25°C) for efficient extraction of ccfDNA from plasma. • Extraction of ccfDNA can be performed manually with the QIAamp Circulating Nucleic Acid Kit or automatically using the QIAsymphony PAXgene Blood ccfDNA Kit. • Automated ccfDNA extraction with the QIAsymphony protocol offers minimal variation between replicates, highly reliable results and no cross-contamination. • ccfDNA yield from PAXgene Blood ccfDNA Tubes stored for up to 7 days at room temperature is comparable to unstabilized EDTA blood on day 0. Study 2: • PAXgene Blood ccfDNA System fulfills requirements for whole blood stabilization and ccfDNA purification for NIPT. • PAXgene Blood ccfDNA System is fully compatible with the NGS-based LifeCodexx PrenaTest. PAXgene Blood ccfDNA System Workflow: Integrated Collection-Stabilization-Preparation (CSP) System Sample Collection Preanalytical Workflow QIAsymphony PAXgene Blood ccfDNA Tube Blood collection and stabilization Transport, storage, plasma separation ccfDNA extraction Real-time PCR, NGS, other Data analysis QIAsymphony PAXgene Blood ccfDNA Kit Analytical Assays Insight Study 1: Verification of the PAXgene Blood ccfDNA System Table 1. System performance evaluation of the PAXgene Blood ccfDNA System Study 1A Comparison of PAXgene automated kit vs. manual procedure 1 Study Parameters Samples tested 825 Subjects 189 Runs 42 Instruments 6 Study Results ccfDNA yield 104% Portion of dsDNA 2 > 96% Total failures 0 Study 1B Evaluation of PAXgene system performance 3 Study Parameters Samples tested 1636 Subjects 204 Runs 71 Instruments 6 Study Results Yield compared to EDTA plasma 4 No significant difference ccfDNA stability at room temperature 5 7 days Cross-contamination No evidence Eluate freeze/thaw cycles 3 Sample throughput per day 6 192 Open reagent cartridge stability 6 weeks Runtime for 96 samples 5 h 40 min 1 QIAamp Circulating Nucleic Acid Kit; 2 Tested with restriction enzyme based assay for subset of eluates; QIAamp circulating Nucleic Acid Kit eluates with comparable portion of double-stranded DNA (dsDNA); 3 Performance evaluated against RUO product specifications; 4 Tested with 60 samples from 20 subjects, directly after blood collection; 5 Tested with 60 samples from 20 subjects; 6 Includes plasma processing and overnight runs Shipment and Storage Simulation 0.0 0.5 1.0 1.5 2.0 2.5 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 Avg. Relative Yield Compared to Day 0 Sample Number Relative yield at day 3 Relative yield at day 7 Figure 2. Relative ccfDNA yield compared to eluates generated from plasma separated directly after blood collection. Plasma was generated from blood collected in PAXgene Blood ccfDNA Tubes after 5 hours of agitation (5 rpm) and 3 or 7 days of horizontal storage at 25°C to simulate typical transport and storage conditions. ccfDNA was purified manually using the QIAamp Circulating Nucleic Acid Kit. Acceptance criteria: Relative yield must be ≥ 0.5 and ≤ 2.0 compared to Day 0, which equals, including the assay’s imprecision [dotted line], a relative yield of ≥ 0.41 and ≤ 2.41. y = 1.11x - 1.85 R² = 0.91 15 16 17 18 19 20 15 16 17 18 19 20 PAXgene Blood ccfDNA Tube C T Values EDTA Tube C T Values Relative ccfDNA Yield Manual Sample Preparation y = 0.94x + 1.37 R² = 0.91 17 18 19 20 21 22 17 18 19 20 21 22 PAXgene Blood ccfDNA Tube C T Values EDTA Tube C T Values Relative ccfDNA Yield Automated Sample Preparation A. B. Figure 3. Relative ccfDNA yield of paired plasma samples collected in PAXgene Blood ccfDNA Tubes as compared to EDTA tubes. C T values of a validated 18S rDNA PCR assay (precision range ±3σ = 0.27 ΔC T indicated as dotted lines) are shown in a scatter plot. ccfDNA was either isolated from plasma manually with the QIAGEN QIAamp Circulating Nucleic Acid Kit (n = 60) (A) or with the QIAsymphony (QS) SP instrument using the QIAsymphony PAXgene Blood ccfDNA Kit and 4.8 ml plasma protocol for PAXgene Blood ccfDNA Tubes or the QIAGEN QIAsymphony Circulating DNA Kit for EDTA tubes (n = 180) (B). Instrument 1 Instrument 2 Instrument 3 Batch 1 neg. 22.11 neg. 22.44 neg. 23.45 Batch 1 neg. 22.44 neg. 22.45 neg. 22.65 Batch 1 neg. 22.27 neg. 22.28 neg. 22.28 22.69 neg. 22.6 neg. 22.35 neg. 22.73 neg. 22.42 neg. 22.13 neg. 22.64 neg. 22.21 neg. 22.37 neg. neg. 22.14 neg. 22.66 neg. 23.27 neg. 22.38 neg. 22.38 neg. 22.48 neg. 22.36 neg. 22.34 neg. 22.25 22.47 neg. 22.48 neg. 22.4 neg. 22.51 neg. 22.38 neg. 23.26 neg. 22.46 neg. 22.13 neg. 22.38 neg. Batch 2 neg. neg. neg. neg. neg. neg. Batch 2 neg. neg. neg. neg. neg. neg. Batch 2 neg. neg. neg. neg. neg. neg. neg. neg. neg. neg. neg. neg. neg. neg. neg. neg. neg. neg. neg. neg. neg. neg. neg. neg. neg. neg. neg. neg. neg. neg. neg. neg. neg. neg. neg. neg. neg. neg. neg. neg. neg. neg. neg. neg. neg. neg. neg. neg. neg. neg. neg. neg. neg. neg. neg. neg. neg. neg. neg. neg. Batch 3 22.31 neg. 22.35 neg. 22.42 neg. Batch 3 22.61 neg. 22.35 neg. 22.34 neg. Batch 3 22.38 neg. 22.29 neg. 22.21 neg. neg. 22.16 neg. 22.17 neg. 22.14 neg. 22.04 neg. 22.42 neg. 22.33 neg. 22.25 neg. 22.21 neg. 22.34 22.37 neg. 22.34 neg. 22.08 neg. 23.33 neg. 22.58 neg. 22.51 neg. 22.48 neg. 22.32 neg. 22.37 neg. neg. 22.15 neg. 30.18 neg. 22.14 neg. 22.24 neg. 22.54 neg. 22.27 neg. 22.23 neg. 22.23 neg. 22.36 Batch 4 neg. neg. neg. neg. neg. neg. Batch 4 neg. neg. neg. neg. neg. neg. Batch 4 neg. neg. neg. neg. neg. neg. neg. neg. neg. neg. neg. neg. neg. neg. neg. neg. neg. neg. neg. neg. neg. neg. neg. neg. neg. neg. neg. neg. neg. neg. neg. neg. neg. neg. neg. neg. neg. neg. neg. neg. neg. neg. neg. neg. neg. neg. neg. neg. neg. neg. neg. neg. neg. neg. neg. neg. neg. neg. neg. neg. Figure 4: Cross Contamination Assessment. C T values of a validated Y-chromosomal DYS14 PCR assay are shown. Three cross contamination test runs of 96 blood samples collected into PAXgene Blood ccfDNA Tubes, were carried out on 3 different QIAsymphony SP instruments using the QIAsymphony PAXgene kit and LAF protocol with 4.8 ml plasma. Per run 24 DYS14-positive (male) and 72 DYS14-negative (female) plasma samples were processed in a checkerboard setup interrupted by negative batches to test for batch to batch contamination.
Transcript of Integrated Workflow for Blood Collection, ccfDNA Stabilization … · 2020-01-05 · Integrated...
Integrated Workflow for Blood Collection, ccfDNA Stabilization and Extraction for Non-Invasive Prenatal Testing1Andrea Ullius, 1Thorsten Voss, 2Stephan Busche, 2Wera Hofmann, 1Daniel Grölz 1PreAnalytiX GmbH, Hilden, Germany; 2LifeCodexx, Konstanz, Germany
BackgroundNon-invasive prenatal testing (NIPT) uses fetal circulating cell-free DNA (ccfDNA) in the maternal blood circulation. The release of maternal genomic DNA from blood cells during blood collection, transport and storage significantly reduces the sensitivity of the next-generation sequencing (NGS) based NIPT assays. Moreover, the reliable and reproducible extraction of the low abundance and short fetal ccfDNA fragments is technically challenging. Both difficulties are addressed by the PAXgene® Blood ccfDNA System*, which consists of a blood collection tube (PAXgene Blood ccfDNA Tube*) and a ccfDNA purification kit (QIAsymphony® PAXgene Blood ccfDNA Kit*). The PAXgene Blood ccfDNA Tube uses a plastic BD Vacutainer® tube with a unique, non-crosslinking chemistry for stabilization of blood cells. The QIAsymphony PAXgene Blood ccfDNA Kit can be used for automated isolation of either small ccfDNA fragments or co-isolation of small and large ccfDNA fragments. The performance of the new system was evaluated in two research studies in comparison to K2EDTA tubes and the Streck Cell-Free DNA BCT®.
PAXgene Blood ccfDNA Tube (RUO)
Tube features:• Effective stabilization of:
– White blood cells – Helps prevent release of gDNA
– Red blood cells – Helps minimize hemolysis
• Helps maximize ccfDNA yield from plasma, minimize background gDNA
• Non-crosslinking, no DNA modification
• BD Vacutainer plastic tube with BD Hemogard™ safety closure – Helps minimize risk of tube breakage – Enhances safety for healthcare and lab
personnel – Helps minimize contamination between samples – Consistent blood draw volume
QIAsymphony PAXgene Blood ccfDNA Kit (RUO)
Kit features:• Dedicated ccfDNA isolation technology for use with
the PAXgene Blood ccfDNA Tube – Binding chemistry optimized for use with
PAXgene Blood ccfDNA Tube reagent – Optimized input volumes to accommodate
higher volume plasma
• Two protocol lines – Standard protocol for preferred extraction
of small fragments (≤ 500 bp) with 60 µl elution volume
– Large fragment protocol for co-isolation of large fragments (> 500 bp) with flexible elution volume (60, 100, 150 µl)
• 2.4 ml or 4.8 ml plasma processing
• Prefilled cartridges are ready to use
QIAsymphony PAXgene Blood ccfDNA Protocols
Standard Protocol Line (STA)(small fragments preferred)
2.4 or 4.8 ml plasma protocol
Large Fragment Protocol Line (LAF)(efficient co-isolation of large fragments)
2.4 or 4.8 ml plasma protocol
Binding/proteinase K digestion with Buffer BCD1
Binding/proteinase K digestion with Buffer BCD2
Wash 1
Wash 2
Wash 3
Elution60 µl
Elution60/100/150 µl
Wash 1
Wash 2
Wash 3
Whole blood collected, transported and stored in PAXgene Blood ccfDNA Tubes
Plasma generation
Automated ccfDNA isolation
Figure 1. Standard and large fragment protocol lines. With the standard protocols (STA) predominately small ccfDNA fragments are isolated while large fragment protocols (LAF) efficiently co-purify large and small ccfDNA fragments. Both protocol lines consist of protocols for 2.4 ml or 4.8 ml plasma input.
Disclaimer*For Research Use Only. Not for use in diagnostic procedures. For up-to-date licensing information and product-specific disclaimers, see the respective PreAnalytiX or QIAGEN handbook or user manual.
PreAnalytiX or QIAGEN kit handbooks and user manuals are available at www.preanalytix.com or www.qiagen.com or can be requested from QIAGEN Technical Services or your local distributor.
Trademarks: QIAGEN®, QIAamp®, QIAsymphony® (QIAGEN Group); PAXgene® (PreAnalytiX GmbH), BD Hemogard™, BD Vacutainer® (Becton, Dickinson and Company), PrenaTest®, QuantYfeX® (LifeCodexx AG); HiSeq™ (Illumina, Inc.); Cell-Free DNA BCT® (Streck, Inc.). Registered names, trademarks, etc. used in this document, even when not specifically marked as such, are not to be considered unprotected by law.
© 2016 PreAnalytiX GmbH, all rights reserved.
Study 2: Validation of the PAXgene Blood ccfDNA System for non-invasive prenatal testing (NIPT) application
Study Setup:
• Blood samples were collected from 62 consented pregnant women
• Two blood collection tubes were collected per patient: – Tube 1: Streck Cell-Free DNA BCT – Tube 2: PAXgene Blood ccfDNA Tube
• Plasma processing from half of the blood samples collected into PAXgene tubes was performed with one centrifugation step, the remainder with two centrifugation steps
• All samples underwent the regular PrenaTest procedure per the standard assay protocol
• 25 plasma samples collected into PAXgene Blood ccfDNA Tubes were selected for downstream analysis (12 with one, 13 with two centrifugation steps)
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QuantYfeX: Relative Fetal ccfDNA Content
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Figure 5: Relative fetal fraction of paired plasma samples collected in PAXgene Blood ccfDNA Tubes or Streck Cell-Free DNA BCT tubes.
Relative amount of fetal ccfDNA in maternal background was quantified with methylation sensitive QuantYfeX QC Assay. Relative fetal ccfDNA fractions (PAXgene median: 13.7%; Streck BCT median: 12.4%) in maternal blood were not significantly different between the two different tubes (p-value: 0.20 paired T-Test, two-tailed distribution).
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Figure 6: NGS read out quality for paired plasma samples collected in PAXgene Blood ccfDNA Tubes or Streck Cell-Free DNA BCT tubes.
ccfDNA from Streck BCTs and PAXgene tubes sequenced on Illumina HiSeq™ with NEB library preparation. There were significantly more mapped reads with ccfDNA from PAXgene tubes (p-value: 1.78E-04, two-tailed paired T-test; A).
There was no significant difference in unique mapped reads (B).
Study 2 Results1. Relative fetal ccfDNA fractions in maternal blood in both tubes were not significantly different
2. Significantly more mapped reads with ccfDNA from PAXgene tubes
3. No significant difference in uniquely mapped reads
4. No significant difference with PAXgene tube samples processed with one or two centrifugation steps
5. All samples which passed the internal quality standards produced equivalent results with the PrenaTest assay
ConclusionsStudy 1:
• PAXgene Blood ccfDNA Tubes stabilize whole blood samples for up to 7 days of storage at room temperature (15–25°C) for efficient extraction of ccfDNA from plasma.
• Extraction of ccfDNA can be performed manually with the QIAamp Circulating Nucleic Acid Kit or automatically using the QIAsymphony PAXgene Blood ccfDNA Kit.
• Automated ccfDNA extraction with the QIAsymphony protocol offers minimal variation between replicates, highly reliable results and no cross-contamination.
• ccfDNA yield from PAXgene Blood ccfDNA Tubes stored for up to 7 days at room temperature is comparable to unstabilized EDTA blood on day 0.
Study 2:
• PAXgene Blood ccfDNA System fulfills requirements for whole blood stabilization and ccfDNA purification for NIPT.
• PAXgene Blood ccfDNA System is fully compatible with the NGS-based LifeCodexx PrenaTest.
PAXgene Blood ccfDNA System Workflow: Integrated Collection-Stabilization-Preparation (CSP) System
Sample Collection Preanalytical Workflow
QIAsymphony
PAXgene Blood ccfDNA Tube
Blood collectionand stabilization
Transport, storage,plasma separation ccfDNA extraction Real-time PCR,
NGS, other Data analysis
QIAsymphony PAXgene Blood ccfDNA Kit
Analytical Assays Insight
Study 1: Verification of the PAXgene Blood ccfDNA System
Table 1. System performance evaluation of the PAXgene Blood ccfDNA System
Study 1AComparison of PAXgene automated kit vs. manual procedure1
Study ParametersSamples tested 825Subjects 189Runs 42Instruments 6Study ResultsccfDNA yield 104%Portion of dsDNA2 > 96%Total failures 0
Study 1BEvaluation of PAXgene system performance3
Study ParametersSamples tested 1636Subjects 204Runs 71Instruments 6Study ResultsYield compared to EDTA plasma4 No significant differenceccfDNA stability at room temperature5 7 daysCross-contamination No evidenceEluate freeze/thaw cycles 3Sample throughput per day6 192Open reagent cartridge stability 6 weeksRuntime for 96 samples 5 h 40 min1QIAamp Circulating Nucleic Acid Kit; 2Tested with restriction enzyme based assay for subset of eluates; QIAamp circulating Nucleic Acid Kit eluates with comparable portion of double-stranded DNA (dsDNA); 3Performance evaluated against RUO product specifications; 4Tested with 60 samples from 20 subjects, directly after blood collection; 5Tested with 60 samples from 20 subjects; 6Includes plasma processing and overnight runs
Shipment and Storage Simulation
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Relative yield at day 3 Relative yield at day 7
Figure 2. Relative ccfDNA yield compared to eluates generated from plasma separated directly after blood collection.
Plasma was generated from blood collected in PAXgene Blood ccfDNA Tubes after 5 hours of agitation (5 rpm) and 3 or 7 days of horizontal storage at 25°C to simulate typical transport and storage conditions. ccfDNA was purified manually using the QIAamp Circulating Nucleic Acid Kit.
Acceptance criteria: Relative yield must be ≥ 0.5 and ≤ 2.0 compared to Day 0, which equals, including the assay’s imprecision [dotted line], a relative yield of ≥ 0.41 and ≤ 2.41.
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Figure 3. Relative ccfDNA yield of paired plasma samples collected in PAXgene Blood ccfDNA Tubes as compared to EDTA tubes.
CT values of a validated 18S rDNA PCR assay (precision range ±3σ = 0.27 ΔCT indicated as dotted lines) are shown in a scatter plot. ccfDNA was either isolated from plasma manually with the QIAGEN QIAamp Circulating Nucleic Acid Kit (n = 60) (A) or with the QIAsymphony (QS) SP instrument using the QIAsymphony PAXgene Blood ccfDNA Kit and 4.8 ml plasma protocol for PAXgene Blood ccfDNA Tubes or the QIAGEN QIAsymphony Circulating DNA Kit for EDTA tubes (n = 180) (B).
Instrument 1 Instrument 2 Instrument 3
Batch 1 neg. 22.11 neg. 22.44 neg. 23.45 Batch 1 neg. 22.44 neg. 22.45 neg. 22.65 Batch 1 neg. 22.27 neg. 22.28 neg. 22.28
22.69 neg. 22.6 neg. 22.35 neg. 22.73 neg. 22.42 neg. 22.13 neg. 22.64 neg. 22.21 neg. 22.37 neg.
neg. 22.14 neg. 22.66 neg. 23.27 neg. 22.38 neg. 22.38 neg. 22.48 neg. 22.36 neg. 22.34 neg. 22.25
22.47 neg. 22.48 neg. 22.4 neg. 22.51 neg. 22.38 neg. 23.26 neg. 22.46 neg. 22.13 neg. 22.38 neg.
Batch 2 neg. neg. neg. neg. neg. neg. Batch 2 neg. neg. neg. neg. neg. neg. Batch 2 neg. neg. neg. neg. neg. neg.
neg. neg. neg. neg. neg. neg. neg. neg. neg. neg. neg. neg. neg. neg. neg. neg. neg. neg.
neg. neg. neg. neg. neg. neg. neg. neg. neg. neg. neg. neg. neg. neg. neg. neg. neg. neg.
neg. neg. neg. neg. neg. neg. neg. neg. neg. neg. neg. neg. neg. neg. neg. neg. neg. neg.
Batch 3 22.31 neg. 22.35 neg. 22.42 neg. Batch 3 22.61 neg. 22.35 neg. 22.34 neg. Batch 3 22.38 neg. 22.29 neg. 22.21 neg.
neg. 22.16 neg. 22.17 neg. 22.14 neg. 22.04 neg. 22.42 neg. 22.33 neg. 22.25 neg. 22.21 neg. 22.34
22.37 neg. 22.34 neg. 22.08 neg. 23.33 neg. 22.58 neg. 22.51 neg. 22.48 neg. 22.32 neg. 22.37 neg.
neg. 22.15 neg. 30.18 neg. 22.14 neg. 22.24 neg. 22.54 neg. 22.27 neg. 22.23 neg. 22.23 neg. 22.36
Batch 4 neg. neg. neg. neg. neg. neg. Batch 4 neg. neg. neg. neg. neg. neg. Batch 4 neg. neg. neg. neg. neg. neg.
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neg. neg. neg. neg. neg. neg. neg. neg. neg. neg. neg. neg. neg. neg. neg. neg. neg. neg.
Figure 4: Cross Contamination Assessment.
CT values of a validated Y-chromosomal DYS14 PCR assay are shown. Three cross contamination test runs of 96 blood samples collected into PAXgene Blood ccfDNA Tubes, were carried out on 3 different QIAsymphony SP instruments using the QIAsymphony PAXgene kit and LAF protocol with 4.8 ml plasma. Per run 24 DYS14-positive (male) and 72 DYS14-negative (female) plasma samples were processed in a checkerboard setup interrupted by negative batches to test for batch to batch contamination.
