QIAsymphony PAXgene Blood ccfDNA Kit

For purification of ccfDNA from plasma using the QIAsymphony SP

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QIAsymphony PAXgene Blood ccfDNA Kit (192)

Cat. No. / ID: 768536

Reagent cartridges, accessories and proteinase K vials for 192 preps

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.

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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

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

High performance, comparable to QIAamp Circulating Nucleic Acid Kit.

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 ). 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 ).

See figures

PCR results from blood collected in PAXgene Blood ccfDNA Tubes remain stable over storage time.

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 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

The QIAsymphony SP processes samples containing magnetic particles.

Two protocol lines of the QIAsymphony PAXgene Blood ccfDNA Kit for different research needs.

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).

Specifications

Features	Specifications
Applications	Wide spectrum of research applications, including analysis of cancer tumor DNA and non-invasive prenatal testing
Format	Magnetic beads
Input volume	2.4 ml or 4.8 ml plasma
Throughput	192 samples per working day and instrument
Time per run	96 samples of 4.8 ml plasma in 5 h 40 min
Processing	Manual: plasma centrifugation Automated: QIAsymphony SP
Shelf life	6 weeks for an open reagent cartridge
Elution volume	60 µl for the STA protocol line 60, 100 or 150 µl for the LAF protocol line

Resources

Download Safety Data Sheets for QIAGEN product components.

Ullius et al., 2015

Ullius et al., 2016

Ullius et al.

Voss et al., 2017

For purification of ccfDNA from human whole blood stabilized in PAXgene Blood ccfDNA Tubes using the QIAsymphony SP and QIAsymphony PAXgene Blood ccfDNA Kit.

For purification of ccfDNA from plasma using the QIAsymphony SP

For use with software version 5.0 or higher

FAQ

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

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

The expiration date is printed on the kit label and encoded in the bar code of the reagent cartridges.

FAQ ID - 3651

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

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

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

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

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

FAQ ID - 3636

The expiration date is printed on the tube label and on the shelf carton.

FAQ ID - 3633

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

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

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

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

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

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

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

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

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

Yes, as long as the phlebotomy instructions included with the PAXgene Blood ccfDNA Tubes are properly followed.

FAQ ID - 3630

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

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

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

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

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

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

Eluates should be stored at −20°C (long-term storage studies are ongoing).

FAQ ID - 3657

Yes, a customized protocol for elution with 50 µl is available on request.

FAQ ID - 3656

Whole blood drawn into PAXgene Blood ccfDNA Tubes can be stored at 15–25°C for up to 7 days.

FAQ ID - 3639

The normal protocols enable processing 2.4 or 4.8 ml plasma. Other volumes can be accommodated using customized protocols.

FAQ ID - 3648

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

FAQ ID - 3634

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