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QIAsymphony PAXgene Blood ccfDNA Kit

For purification of ccfDNA from plasma using the QIAGEN QIAsymphony SP instrument

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

 

Feature Specification
Format Magnetic beads
Technology Magnetic particle technology
Sample input volume 2.4 ml or 4.8 ml plasma
Elution volume 60 µl for the STA protocol line
60, 100 or 150 µl for the LAF protocol line
Sample type Plasma from whole blood collected into PAXgene Blood ccfDNA Tubes
Time per run 96 samples with 4.8 ml plasma in < 5 h 40 m
Throughput 192 samples per working day and instrument
Processing Automated: QIAGEN QIAsymphony SP Instrument
Shelf life of open reagent cartridge 12 weeks
Processing Manual: centrifugation

 

Intended use

The PAXgene Blood ccfDNA System consists of a blood collection tube (PAXgene Blood ccfDNA Tube) and a ccfDNA purification kit for the QIAsymphony SP (QIAsymphony PAXgene Blood ccfDNA Kit). The PAXgene Blood ccfDNA System is intended for the collection, storage and transport of blood, the stabilization of ccfDNA in a closed tube, and the subsequent isolation and purification of ccfDNA from plasma for research applications.

The QIAsymphony PAXgene Blood ccfDNA Kit uses magnetic particle technology for automated isolation and purification of ccfDNA from plasma generated from whole blood collected into PAXgene Blood ccfDNA Tubes.

For Research Use Only. Not for use in diagnostic procedures. The performance characteristics of this product have not been fully established.

Workflow of ccfDNA isolation
Two protocol lines of the QIAsymphony PAXgene Blood ccfDNA Kit for different research needs.
With the standard protocols (STA), predominantly small ccfDNA fragments are isolated. The large fragment protocols (LAF) efficiently co-purify large and small ccfDNA fragments. Both protocol lines [...]
Performance analysis of ccfDNA isolation
High performance, comparable to the QIAamp Circulating Nucleic Acid Kit.
Results from two studies assessing the performance of the QIAsymphony PAXgene Blood ccfDNA Kit. Study A compares ccfDNA yield and portion of dsDNA of eluates generated with the QIAsymphony PAXgene [...]
Magnetic-bead technology of the QIAsymphony
The QIAsymphony SP processes samples containing magnetic particles.
A magnetic rod protected by a rod cover enters a well containing the sample and attracts the magnetic particles. The magnetic rod cover is positioned above another well and the magnetic particles are [...]
Stable results over storage time
PCR results from blood collected in PAXgene Blood ccfDNA Tubes remain stable over storage time.
The change in ΔCT values of target versus control signal for ccfDNA isolated from whole blood stored in EDTA and PAXgene Blood ccfDNA Tubes was measured at different storage intervals. Shown are the [...]
Accuracy of results in methylation assays
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, [...]
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Performance

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

 

Principle

Apoptosis and lysis of white blood cells in unstabilized whole blood releases intracellular DNA into plasma, which dilutes naturally occurring ccfDNA. PAXgene Blood ccfDNA Tubes contain an additive that anticoagulates blood and stabilizes blood cells via a non-crosslinking stabilization solution. This helps prevent the release of intracellular DNA and maintains constant ccfDNA levels during sample transport and storage prior to processing (see PCR results from blood collected in PAXgene Blood ccfDNA Tubes remain stable over storage time). 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 Isolated ccfDNA is compatible with methylation-specific downstream applications).

 

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 further ensure separation of plasma from cellular debris. The purified plasma is then transferred to a third tube which is then 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 The QIAsymphony SP processes samples containing magnetic particles). Depending on the chosen protocol line, predominantly small ccfDNA fragments or small and large ccfDNA fragments are isolated (see Two protocol lines of the QIAsymphony PAXgene Blood ccfDNA Kit for different research needs). Three wash steps ensure contaminant removal. Finally, ccfDNA is eluted from the magnetic particles and is ready for use in downstream applications.

 

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

Handbooks (1)

File sizeFormatDownload
QIAsymphony PAXgene Blood ccfDNA Kit Handbook 1178 KB pdf

Brochures (2)

File sizeFormatDownload
Save time and resources by processing PAXgene Blood ccfDNA Tubes directly on the QIAsymphony SP 926 KB pdf
PAXgene Blood ccfDNA System Brochure 1640 KB pdf

Technical information (1)

File sizeFormatDownload
PAXgene Blood ccfDNA System Primary Tube Handling: Shortened workflow processing PAXgene Blood ccfDNA Tubes directly with the QIAsymphony PAXgene Blood ccfDNA Kit 873 KB pdf

Posters (9)

File sizeFormatDownload
Analytical Validation of a Circulating Tumor Methylated-DNA Assay for Detection of Colorectal Cancer Recurrence; Alsobrook et al., AMP 2017 824 KB pdf
Performance Evaluation of Collection/Stabilization/Purification Systems for Liquid Biopsy Cancer Biomarker Applications; Groelz et al., AMP 2017 7455 KB pdf
Verification of Liquid Biopsy Blood Collection, ccfDNA Stabilization and Purification Systems for Liquid Biopsy Cancer Biomarker Applications; Groelz et al., CNAPS 2017 4105 KB pdf
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; Voss et al., CNAPS 2017 4377 KB pdf
Evaluation of a New System for Collection, Stabilization, and Purification of Circulating Tumor DNA; Fleischhacker et al., AACR 2017 6839 KB pdf
Integrated Workflow for Blood Collection, ccfDNA Stabilization and Extraction for Non-invasive Prenatal Testing. Ullius et al., AMP 2016 7566 KB pdf
The PAXgene Blood ccfDNA System: An Integrated System for Collection, Stabilization and Purification of Circulating Tumor DNA. Fleischhacker et al., AMP 2016 5343 KB pdf
Performance evaluation of a new integrated blood collection/sample preparation system for the stabilization and extraction of circulating cell-free DNA (ccfDNA); Ullius et al., EACR 2015 5286 KB pdf
A new blood collection tube and automated extraction method for stabilization and analysis of ccfDNA; Ulius et al., 2015 3250 KB pdf

References (2)

File sizeFormatDownload
Diagnostics using cell-free circulating DNA from the blood; Voss T; Article translated from german after publication in Blutbild, Issue 31, May 2017 by BD 236 KB pdf
Circulating Cell-Free DNA Pre-analytics: The Importance of Standardized Workflows for Liquid Biopsy Applications 1210 KB pdf

FAQ (31)

    Sample Collection and Phlebotomy

    1. 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.

    2. 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.

    3. 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 ito the patient's arm.

    4. 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.

    5. What is the blood draw volume of the PAXgene Blood ccfDNA Tube?

    The blood draw volume is 10 ml and the stabilzation regent volume is 1.5 ml. Thus, a filled tube holds a volume of 11.5 ml.

    6. 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 under the Resources tab for more details. Additional information can be found in the Safety Data Sheet, under the Resources Tab.

    7. 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.

    8. How should unused PAXgene Blood ccfDNA Tubes be stored?

    Store unused PAXgene Blood ccfDNA Tubes at room temperature (15–25°C) and protect them from direct sunlight.

    9. 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. Ensure that blood has stopped flowing into the tube before removing it from the holder.

    For more information on phlebotomy technique, please refer to the PAXgene Blood ccfDNA Tube Product Circular under the Resources tab.

    10. Are PAXgene Blood ccfDNA Tubes sterile?

    Yes, the inside of PAXgene Blood ccfDNA Tubes is sterilized during production.

    11. What is the expected ccfDNA yield from plasma generated from 10 ml 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.

    12. Can I use plasma from underfilled PAXgene Blood ccf DNA 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.

    Sample Storage and Archiving

    1. 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.

    2. 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.

    3. 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.

    4. 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.

    Plasma processing

    1. Is the 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.

    2. 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).

    3. How should I thaw frozen plasma samples?

    Thaw plasma at room temperature (15–25°C). To minimize the formation of cryoprecipitates, do not thaw plasma at lower temperatures (e.g., 4°C). Alternatively, thaw plasma for 30 minutes at 30°C to completely avoid the formation of cryoprecipitates.

    4. What should I do if cryoprecipitates form while thawing a frozen plasma sample?

    If cryoprecipitates form in the thawed plasma, vortex the tube for 30 seconds and proceed according to the instructions provided with the QIAamp Circulating Nucleic Acid Kit or the QIAsymphony PAXgene Blood ccfDNA Kit.

    Do not centrifuge the plasma to remove cryoprecipitates because they may contain ccfDNA.

    ccfDNA Isolation and Processing

    1. 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.

    2. 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.

    3. 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 use.

    4. 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.

    5. 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.

    6. 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).

    7. Which protocol line should I use to process plasma samples?

    The QIAsymphony PAXgene Blood ccfDNA Kit can be used with 2 protocol lines. The standard protocols (STA) predominantly 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.

    8. 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.

    9. Does the QIAsymphony PAXgene Blood ccfDNA Kit also allow eluting ccfDNA in less than 60 µl?

    Yes, a customized protocol for elution in 50 µl is available upon request.

    10. 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).

    Downstream Applications

    1. 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.

ProductCatalog Nr.Price (USD)

QIAsymphony PAXgene Blood ccfDNA Kit (192)

Reagent cartridges, accessories and proteinase K vials for 192 preps.
7685363886.00