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QIAsymphony PAXgene Blood ccfDNA Kit

For purification of ccfDNA from plasma using the QIAsymphony SP

Features

  • Optimized chemistry and input volumes for PAXgene Blood ccfDNA Tubes
  • Separate protocol lines for small (STA) and large (LAF) ccfDNA fragments
  • Prefilled reagent cartrdiges with bar code for safety and ease of use
  • Standardized processing for high reproducibility

Useful Resources

QIAsymphony PAXgene Blood ccfDNA Kit Handbook QIAsymphony Protocol Sheet PAXgene ccfDNA_2400 and ccfDNA_4800 A New Blood Collection Tube and Automated Extraction Method for Stabilization and Analysis of ccfDNA
S_1084_5_GEN_disclaimer
Want to try this solution for the first time?
Get in touch with our specialists if you need a quote or if you would like to discuss your project.
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QIAsymphony PAXgene Blood ccfDNA Kit (192)

Cat. No. / ID: 768536

Reagent cartridges, accessories and proteinase K vials for 192 preps
 $4,712.00
For Research Use Only. Not for use in diagnostics procedures. No claim or representation is intended to provide information for the diagnosis, prevention, or treatment of a disease.
Want to try this solution for the first time?
Get in touch with our specialists if you need a quote or if you would like to discuss your project.
CONTACT A SPECIALIST

Product Details


The PAXgene Blood ccfDNA System consists of a blood collection tube (PAXgene Blood ccfDNA Tube) and a ccfDNA purification kit for the QIAsymphony (QIAsymphony PAXgene Blood ccfDNA Kit).

Performance

The fully automated QIAsymphony PAXgene Blood ccfDNA Kit procedure generates ccfDNA yields comparable to the manual QIAamp Circulating Nucleic Acid Kit procedure (see figure  High performance, comparable to QIAamp Circulating Nucleic Acid Kit). Since yield is strongly dependent of donor, individual yields may vary. Onboard measures of the QIAsymphony SP minimize cross-contamination.

See figures
T_0018_QS_PX_ccfDNA_performanceT_0018_QS_PX_ccfDNA_performance

Principle

Apoptosis and lysis of white blood cells in unstabilized whole blood releases genomic DNA into plasma, which dilutes naturally occurring ccfDNA. PAXgene Blood ccfDNA Tubes contain an additive that prevents blood coagulation and immediately stabilizes blood cells via a non-crosslinking stabilization solution (see figure  PCR results from blood collected in PAXgene Blood ccfDNA Tubes remain stable over storage time). This impedes the release of intracellular DNA and maintains constant ccfDNA levels in plasma. When used with PAXgene Blood ccfDNA Tubes, the proven magnetic particle technology of the QIAsymphony PAXgene Blood ccfDNA Kit provides a rapid, automated procedure to isolate ccfDNA of high quality and purity (see figure  Isolated ccfDNA is compatible with methylation-specific downstream applications).
See figures
LG_0323_QS_PX_stabilityLG_0323_QS_PX_stabilityBC_0732_QS_PX_ccfDNA_accuracyBC_0732_QS_PX_ccfDNA_accuracy

Procedure

The workflow begins with a centrifugation step that separates plasma from the cellular fraction of the whole blood collected into PAXgene Blood ccfDNA Tubes. The plasma is then carefully transferred into a second tube. A second centrifugation step can be carried out optionally to remove any traces of buffy coat. The pure plasma is then transferred to a third tube which is loaded into the QIAsymphony SP.
In the QIAsymphony SP, plasma proteins are digested by proteinase K while ccfDNA binds to the surface of magnetic beads (see figure  The QIAsymphony SP processes samples containing magnetic particles). Depending on the chosen protocol line, predominantly small ccfDNA fragments (STA protocol line) or small and large ccfDNA fragments (LAF protocol line) are isolated. Three wash steps ensure contaminant removal. Finally, ccfDNA is eluted from the magnetic particles and is ready for use in downstream applications (see figure  Two protocol lines of the QIAsymphony PAXgene Blood ccfDNA Kit for different research needs).

See figures
QS_ILLU_0038_BioSprint_newQS_ILLU_0038_BioSprint_newFC_0359_QS_PX_ccfDNA_WFFC_0359_QS_PX_ccfDNA_WF

Applications

ccfDNA purified using the QIAsymphony PAXgene Blood ccfDNA Kit and the QIAsymphony SP is ready for use in a wide range of downstream applications, including:

  • PCR, digital, multiplex and quantitative real-time PCR
  • Pharmacogenomic studies
  • SNP genotyping
  • Next generation sequencing
  • Methylation assays

Supporting data and figures

Isolated ccfDNA is compatible with methylation-specific downstream applications.

Whole blood of 4 healthy donors was collected into either EDTA or PAXgene Blood ccfDNA Tubes. Blood was left at room temperature for up to 7 days. Each day, ccfDNA was isolated from samples, bisulphite converted (Epitect FAST kit, QIAGEN) and analyzed with the GEMINI assay (Clinical Genomics) based on 2 methylated (BCAT1 and IKZF1) and 1 control (ACTB) target. Three PCR replicates per target sequence were analyzed. ccfDNA from blood stored in EDTA tubes generated more false positives than from blood stored in PAXgene Blood ccfDNA Tubes (data courtesy of Clinical Genomics).

Isolated ccfDNA is compatible with methylation-specific downstream applications.
Isolated ccfDNA is compatible with methylation-specific downstream applications.
The QIAsymphony SP processes samples containing magnetic particles.
Two protocol lines of the QIAsymphony PAXgene Blood ccfDNA Kit for different research needs.
PCR results from blood collected in PAXgene Blood ccfDNA Tubes remain stable over storage time.
High performance, comparable to QIAamp Circulating Nucleic Acid Kit.

Specifications

FeaturesSpecifications
ApplicationsWide spectrum of research applications, including analysis of cancer tumor DNA and non-invasive prenatal testing
FormatMagnetic beads
Input volume2.4 ml or 4.8 ml plasma
Throughput192 samples per working day and instrument
Time per run96 samples of 4.8 ml plasma in 5 h 40 min
ProcessingManual: plasma centrifugation Automated: QIAsymphony SP
Shelf life6 weeks for an open reagent cartridge
Elution volume60 µl for the STA protocol line 60, 100 or 150 µl for the LAF protocol line

Resources

Safety Data Sheets (1)
Safety Data Sheets
Download Safety Data Sheets for QIAGEN product components.
Scientific Posters (4)
A New Blood Collection Tube and Automated Extraction Method for Stabilization and Analysis of ccfDNA
PDF (3MB)
Ullius et al., 2015

 

Integrated Workflow for Blood Collection, ccfDNA Stabilization and Extraction for Non-invasive Prenatal Testing
PDF (7MB)
Ullius et al., 2016

 

Performance Evaluation of a New Integrated Blood Collection and Sample Preparation System for the Stabilization and Extraction of Circulating Cell-free DNA (ccfDNA)
PDF (5MB)
Ullius et al.

 

A Shortened, Automated Workflow for Processing Plasma Directly From Primary Blood Collection Tubes Reduces Turnaround Time and Risk of Sample Mix-up in ccfDNA Based Applications
PDF (4MB)
Voss et al., 2017

 

Application/Protocol Documents (1)
QIAsymphony Protocol Sheet PAXgene ccfDNA_2400 and ccfDNA_4800
PDF (115KB)
For purification of ccfDNA from human whole blood stabilized in PAXgene Blood ccfDNA Tubes using the QIAsymphony SP and QIAsymphony PAXgene Blood ccfDNA Kit.

 

Kit Handbooks (1)
QIAsymphony PAXgene Blood ccfDNA Kit Handbook
PDF (1MB)
For purification of ccfDNA from plasma using the QIAsymphony SP
Labware Files and Documents (1)
Labware List — QIAsymphony PAXgene Blood ccfDNA Kit (SOW-516-8)
PDF (101KB)
For use with software version 5.0 or higher 
Brochures & Guides (1)
Flyer: Primary Tube Handling with PAXgene Blood ccfDNA System
PDF (2MB)
Application Notes (1)
PAXgene Blood ccfDNA System Primary Tube Handling (EN)
PDF (1MB)

FAQ

Which protocol line should I use to process plasma samples?
The QIAsymphony PAXgene Blood ccfDNA Kit can be sued with 2 protocol lines. The standard protocols (STA) predominantely isolate small ccfDNA fragments. The large fragment protocols (LAF) efficiently co-purify large and small ccfDNA fragments. Both lines include a protocol for 2.4 ml and another for 4.8 ml plasma input.
FAQ ID - 3654
Which elution volumes are available with the QIAsymphony PAXgene Blood ccfDNA Kit?
The elution volume of the standard protocols (STA) is fixed at 60 µl. The large fragment protocols (LAF) enable elution in 3 different volumes: 60, 100 and 150 µl.
FAQ ID - 3655
What is the shelf life of the QIAsymphony PAXgene Blood ccfDNA Kit?
The expiration date is printed on the kit label and encoded in the bar code of the reagent cartridges.
FAQ ID - 3651
Can whole blood in PAXgene Blood ccfDNA Tubes be stored at temperatures above 25°C?
Whole blood drawn into PAXgene Blood ccfDNA Tubes can be stored for at least 24 hours at 35°C. Higher temperatures will lead to lysis of blood cells and release of genomic DNA into plasma.
FAQ ID - 3640
Which downstream applications have been tested with ccfDNA purified from whole blood collected and processed with the PAXgene Blood ccfDNA System?
To date, ccfDNA from blood samples collected and processed using the PAXgene Blood ccfDNA System have been successfully tested in PCR, including multiplex qPCR, and NGS applications.
FAQ ID - 3658
Which tubes are recommended for the QIAsymphony sample rack
The recommended sample input format is the 14 ml Falcon® polystyrene round bottom tube 17 x 100 mm supplied by Corning (formerly supplied by BD).
FAQ ID - 3653
Can whole blood be frozen in PAXgene Blood ccfDNA Tubes?
No. Freezing blood drawn into PAXgene Blood ccfDNA Tubes results in lysis of red and white blood cells and release of genomic DNA.
FAQ ID - 3642
Are PAXgene Blood ccfDNA Tubes sterile?
Yes, the inside of PAXgene Blood ccfDNA Tubes is sterilized during production.
FAQ ID - 3636
What is the shelf life of unused PAXgene Blood ccfDNA Tubes?
The expiration date is printed on the tube label and on the shelf carton. 
FAQ ID - 3633
How long after first use can a reagent cartridge be stored for further use?
When closed properly with the provided strips and stored at room temperature (15–25°C), a reagent cartridge can be used up to 6 weeks after the initial run.
FAQ ID - 3650
Can whole blood in PAXgene Blood ccfDNA Tubes be stored at temperatures below 15°C?
Yes. Whole blood in PAXgene Blood ccfDNA Tubes can be stored at 8–15°C for up to 7 days. Storing blood in PAXgene Blood ccfDNA Tubes below room temperature does not extend stability. 
FAQ ID - 3641
What should I do if cryoprecipitates form while thawing a frozen plasma sample?

If cryoprecipitates cannot be resolved completely by vortexing, the sample should be centrifuged to prevent precipitates from blocking the column. To ensure that no cfDNA is co-precipitated,  centrifugation can be performed at a lower speed (at 6,800xg for 3 minutes at room temperature, as stated in the Protocol Sheet for the QS DSP Circulating DNA Kit). 



FAQ ID - 3646
How long and at which temperature can plasma generated from whole blood in PAXgene Blood ccfDNA Tubes be stored?
Plasma generated from whole blood collected into PAXgene Blood ccfDNA Tubes can be stored at 2–8°C for up to 14 days. We recommend freezing plasma at −20°C or −80°C for longer storage periods (long-term storage studies are ongoing).
FAQ ID - 3644
What is the expected ccfDNA yield from plasma generated from 10 ml of blood?
The yield of ccfDNA depends on the target ccfDNA. Nevertheless, yields obtained from PAXgene Blood ccfDNA Tubes are comparable to those obtained from the same volume of plasma separated directly after drawing blood into a spray-dried EDTA tube. In the PAXgene Blood ccfDNA Tube, however, this yield remains stable for up to 7 days when the tube is stored at 15–25°C or for up to 24 hours when stored at 35°C. In contrast, genomic DNA is released from lysed and apoptotic cells in the unstabilized contents of the EDTA tube soon after blood draw.


FAQ ID - 3637
Do I need to use a blood collection set with the PAXgene Blood ccfDNA Tube?
Yes. Further information on phlebotomy technique with the PAXgene Blood ccfDNA Tube can be found in the PAXgene Blood ccfDNA Tube Product Circular under the Resources tab.
FAQ ID - 3626
How many extractions can I perform with a QIAsymphony PAXgene Blood ccfDNA Kit, using the 2.4 ml or the 4.8 ml protocols?
The number of extractions that can be performed with a kit is independent of sample volume. A kit contains sufficient reagents for 192 extractions.
FAQ ID - 3649
Can reagent from the PAXgene Blood ccfDNA Tube flow back into the patient’s arm?
If the instructions for use are followed, there is no possibility of backflow during phlebotomy and thus, no possibility of reagent going into the patient’s arm.
FAQ ID - 3629
Can I use plasma from underfilled PAXgene Blood ccfDNA Tubes?
A complete 10 ml blood draw ensures optimal results. However, depending on the downstream application and target ccfDNA fragment size, tubes filled with as little as 6.5 ml whole blood can be used without adversely affecting results.
FAQ ID - 3638
Can I use a different brand blood collection set with the PAXgene Blood ccfDNA Tube?
Yes, as long as the phlebotomy instructions included with the PAXgene Blood ccfDNA Tubes are properly followed.
FAQ ID - 3630
What is the reagent in the PAXgene Blood ccfDNA Tube?
The PAXgene Blood ccfDNA Tube contains a proprietary formulation that prevents blood coagulation and the release of genomic DNA from blood cells into plasma. Contents of the tube may cause irritation to the eyes, respiratory system and skin. Please refer to the PAXgene Blood ccfDNA Tube Product Circular. For additional information, visit www.preanalytix.com or review the Safety Data Sheet under the Resources tab.
FAQ ID - 3632
What is the blood draw volume of the PAXgene Blood ccfDNA Tube
The blood draw volume is 10 ml and the stabilization reagent volume is 1.5 ml. Thus, a filled tube holds a volume of 11.5 ml.
FAQ ID - 3631
For what type of sample material is the QIAsymphony PAXgene Blood ccfDNA Kit intended?
The QIAsymphony PAXgene Blood ccfDNA Kit is designed to be used with plasma generated from whole blood collected into PAXgene Blood ccfDNA Tubes.
FAQ ID - 3652
How should I thaw frozen plasma samples?
Thaw the tubes at room temperature (15–25°C). To minimize the formation of cryoprecipitates, do not thaw at lower temperatures (e.g., 4°C). Alternatively, thaw plasma for 30 minutes at 30°C to completely avoid the formation of cryoprecipitates.
FAQ ID - 3645
Are there special considerations for phlebotomy when using the PAXgene Blood ccfDNA Tube (blood collection set, discard tube, positioning of the tube, issues with short draws, etc.)?

Yes. Following a standard phlebotomy protocol, blood is collected into the evacuated tube using a blood collection set and a standardized needle holder that can accommodate a 16 mm diameter tube. Make sure that blood has stopped flowing into the tube before removing the tube from the holder.

For more information on phlebotomy technique, please refer to the PAXgene Blood ccfDNA Tube Product Circular under the Resources tab.
FAQ ID - 3635
Is double centrifugation required for plasma processing?
No. However, a second centrifugation step helps remove traces of buffy coat that may be accidentally transferred when aspirating plasma after the first centrifugation step.
FAQ ID - 3643
How should eluates produced with the PAXgene Blood ccfDNA System be stored?
Eluates should be stored at −20°C (long-term storage studies are ongoing).
FAQ ID - 3657
Does the QIAsymphony PAXgene Blood ccfDNA Kit also allow eluting ccfDNA in less than 60 µl?
Yes, a customized protocol for elution with 50 µl is available on request.
FAQ ID - 3656
How long can whole blood stabilized in PAXgene Blood ccfDNA Tubes be stored at room temperature?
Whole blood drawn into PAXgene Blood ccfDNA Tubes can be stored at 15–25°C for up to 7 days.
FAQ ID - 3639
Which plasma volumes can I process with the QIAsymphony PAXgene Blood ccfDNA protocols?
The normal protocols enable processing 2.4 or 4.8 ml plasma. Other volumes can be accommodated using customized protocols. 
FAQ ID - 3648
How should unused PAXgene Blood ccfDNA Tubes be stored?
Store PAXgene Blood ccfDNA Tubes at room temperature (15–25°C) and protect them from direct sunlight.
FAQ ID - 3634
Why is a blood collection set required to collect blood into the PAXgene Blood ccfDNA Tube?
When used in accordance with instructions, the blood collection set eliminates the possibility of patient contact with the reagent in the tube due to backflow of blood.
FAQ ID - 3628

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