PAXgene Blood ccfDNA Tube
For collection of whole blood samples and stabilization of ccfDNA
- Single tube collection, stabilization, transport and storage
- Stabilization of circulating, cell-free DNA (ccfDNA)
- Sample transport and storage at room temperature for up to 7 days
- Standardized preanalytical processing of samples
|Tube size||16 x 100 mm|
|Blood draw volume||10 ml|
|Additive Volume||1.5 ml|
|Length of stabilization||Up to 7 days at room temperature (15–25°C)
Up to 24 hours at 35°C
|Closure type/color||Pearl white BD Hemogard/Blue stopper|
The PAXgene Blood ccfDNA Tube is a plastic, closed, evacuated tube for the collection, anticoagulation, transport and storage of human whole blood samples and the stabilization of ccfDNA. When the tube is used in conjunction with the QIAsymphony PAXgene Blood ccfDNA Kit or the QIAGEN QIAamp Circulating Nucleic Acid Kit, high quality ccfDNA can be isolated for downstream analytical assays.
For Research Use Only. Not for use in diagnostic procedures. The performance characteristics of this product have not been fully established.
Whole blood was stored in EDTA tubes or PAXgene Blood ccfDNA Tubes. ccfDNA was extracted from the plasma immediately following blood collection (t0) or after 6 days storage (t6) at room temperature. Eluate (1 µl) was analyzed using the Agilent High Sensitivity DNA Kit. Plasma from EDTA tubes showed an increase in apoptotic gDNA fragments, whereas plasma from PAXgene Blood ccfDNA Tubes showed a ccfDNA profile comparable to day 0.
Whole blood from 2 subjects was stored in PAXgene Blood ccfDNA Tubes and EDTA tubes. Genomic DNA was extracted from whole blood samples using the QIAamp Blood Mini Kit immediately after blood collection and after 10 days of storage. Agarose gel electrophoresis was used for size separation of 400 ng extracted gDNA. Fragmentation was evident in the gDNA extracted from whole blood stored for 10 days in EDTA tubes, but was practically absent from the samples stored in PAXgene Blood ccfDNA Tubes.
Whole blood was stored in EDTA tubes or PAXgene Blood ccfDNA Tubes. Hemolysis was determined by measuring the absorption of free hemoglobin in plasma from 6 subjects at 414 nm. Mean absorption and standard deviation are shown. Amount of hemolysis increased with each additional storage time point in the EDTA tubes, but remained relatively constant in the PAXgene Blood ccfDNA additive.
Whole blood from 20 subjects was collected into PAXgene Blood ccfDNA Tubes, EDTA tubes and blood collection tubes designed for ccfDNA stabilization from 2 other suppliers. Plasma was separated directly after blood draw, after 3 days and after 7 days storage at room temperature (15–25°C). Hemolysis was assessed by measuring plasma free hemoglobin (pfHb) using the Catachem Plasma Free Hemoglobin (PFH) Kit on the Beckman Coulter DxC analyzer. Increase in sample hemolysis during sample storage at room temperature was minimized in PAXgene Blood ccfDNA Tubes compared to the other blood collection tubes. This indicates red blood cell lysis and decreased sample integrity in the alternative blood collection tubes.
Whole blood from 8 subjects was collected into EDTA tubes (unstabilized) and PAXgene Blood ccfDNA Tubes (stabilized). Samples were stored for 1 day at 35°C (t1, 35°C), for 6 days at room temperature (t6) or to simulate transport, continually inverted for 5 hours and then stored horizontally for 6 days at room temperature (t6sim). ccfDNA was extracted from plasma using the QIAsymphony SP Protocol for the PAXgene Blood ccfDNA System and ccfDNA yield was quantified by real-time PCR (18S rDNA gene, 66 bp, 180 bp and 500 bp amplicons). Shown is the average ratio of copy numbers at testing time (tx) to copy numbers immediately after blood draw (t0), plus standard deviation. The ratio remained close to 1 in all treatments of the PAXgene Blood ccfDNA stabilized samples, indicating the 18S rDNA copy number did not change over time. A significant increase in 18S rDNA copy number was observed in the EDTA samples after storage, indicating release of gDNA.
PAXgene Blood ccfDNA Tubes simplify the collection and processing of whole blood for subsequent purification of circulating, cell-free DNA (ccfDNA). The tube additive stabilizes blood cells (see PAXgene Blood ccfDNA stabilization helps prevent hemolysis allowing distinct separation of plasma and PAXgene Blood ccfDNA stabilization minimizes hemolysis compared to alternative solutions) and blocks apoptosis (see PAXgene Blood ccfDNA stabilization helps prevent apoptosis and fragmentation of gDNA). Consequently, using PAXgene Blood ccfDNA Tubes for whole blood collection, storage and transport helps prevent the release of intracellular DNA into plasma (see PAXgene Blood ccfDNA stabilization helps prevent release of gDNA into plasma). ccfDNA levels are stable for up to 7 days at room temperature (15-25°C) or up to 24 hours at 35°C, so blood samples can be transported and stored at room temperature to process plasma later (see PAXgene Blood ccfDNA stabilization helps prevent release of gDNA into plasma during storage and transport).
Plasma generated from whole blood collected into PAXgene Blood ccfDNA Tubes and processed with the QIAsymphony PAXgene Blood ccfDNA Kit or the QIAGEN QIAamp Circulating Nucleic Acid Kit, yields high-quality ccfDNA for downstream analytical assays, including but not limited to PCR based assays and Next Generation Sequencing (NGS).
The PAXgene Blood ccfDNA Tube is a plastic, closed, evacuated tube for the collection, anticoagulation, transport and storage of human whole blood samples and the stabilization of ccfDNA. The tube contains an additive that anticoagulates blood and stabilizes blood cells via a non-crosslinking stabilization solution. This helps prevent the release of intracellular DNA into the plasma and maintains constant ccfDNA levels during sample transport and storage before processing.
The PAXgene Blood ccfDNA Tube allows collection of 10 ml whole blood into a closed, evacuated system. Using standard venipuncture technique, blood is collected into an evacuated tube that contains 1.5 ml cell stabilization additive resulting in a ratio of 0.15 ml additive per 1 ml blood. Isolation of ccfDNA from plasma is carried out using the QIAsymphony PAXgene Blood ccfDNA Kit or the QIAGEN QIAamp Circulating Nucleic Acid Kit.
ccfDNA purified from plasma generated from whole blood collected in PAXgene Blood ccfDNA Tubes can be used in a wide range of downstream applications, including:
- PCR, including digital, multiplex and quantitative real-time PCR
- Pharmacogenomic studies
- SNP genotyping
- Next generation sequencing
|Purification of genomic DNA from the PAXgene® Blood ccfDNA Tube using the QIAamp® DNA Blood Mini Kit||518 KB|
|PAXgene Blood ccfDNA System Brochure||1640 KB|
|How to Collect Blood Using the PAXgene Blood ccfDNA Tube||615 KB|
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 blackfow 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.
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 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 protocols. 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).
1. Which downstream applications have been tested with ccfDNA purified from whole blood collected into PAXgene Blood ccfDNA Tubes?
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.
|Product||Catalog Nr.||Price (USD)|
PAXgene Blood ccfDNA Tube (100)100 blood collection tubes (10 ml). To be used in conjunction with QIAsymphony® PAXgene Blood ccfDNA Kit (192) (Cat. no. 768536), QIAGEN QIAamp MinElute ccfDNA Kit (Cat. no. 55204, 55284), or QIAGEN QIAamp® Circulating Nucleic Acid Kit (50) (Cat. no. 55114).