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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

6 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.

Order information

Product

Catalog Nr.

Price

QIAsymphony PAXgene Blood ccfDNA Kit (192)

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

768536
5180 AUD

Details

Performance

The fully automated QIAsymphony PAXgene Blood ccfDNA Kit procedure generates ccfDNA yields comparable to the manual QIAGEN QIAamp Circulating Nucleic Acid Kit procedure (Figure 1). 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 (Figure 2). 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 (Figure 3).

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 (Figure 4). Depending on the chosen protocol line, predominantly small ccfDNA fragments or small and large ccfDNA fragments are isolated (Figure 5). 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
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 Blood ccfDNA Kit to eluates from the manual QIAamp Circulating Nucleic Acid Kit. Study B summarizes performance parameters for the PAXgene ccfDNA system. The QIAsymphony PAXgene Blood ccfDNA Kit provides consistently accurate results. Zoom image

Figure 1. High performance, comparable to the QIAamp Circulating Nucleic Acid Kit.

Results from two studies assessing the performance of the QIAsymphony PAXgene ...
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 relative signals of the 3 mutations covered by the therascreen EGFR assay: (A) point mutation T790M, (B) deletion in exon 19 and (C) L858R. Each PCR was done in duplicate. To calculate the ΔCT value, the average CT of each mutant was subtracted from the average CT of the wildtype sub-assay. The average of all donors is shown in the figure for all test time points. The touchscreen EGFR assay defines a threshold for each sub-assays. ΔCT values above this threshold result in false-negative calls. Results from the ccfDNA isolated from EDTA tubes exceeded this threshold over time whereas results from the PAXgene Blood ccfDNA Tubes remained stable. Zoom image

Figure 2. 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 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). Zoom image

Figure 3. Isolated ccfDNA is compatible with methylation-specific downstream applications.

Whole blood of 4 healthy donors was collected into either EDTA or PAXgene Blood ...
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 released. The QIAsymphony SP uses a magnetic head with an array of 24 magnetic rods, and can therefore process up to 24 samples simultaneously. Collection, transfer and release of magnetic particles is repeated several times during sample processing. Zoom image

Figure 4. The QIAsymphony SP processes samples containing magnetic particles.

A magnetic rod protected by a rod cover enters a well containing the sample and ...
Zoom image

Figure 5. Two protocol lines of the QIAsymphony PAXgene Blood ccfDNA Kit for different research needs.

Resources

QIAsymphony PAXgene Blood ccfDNA Kit

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 into 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 stabilization 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.

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