A Standardized Liquid Biopsy Preanalytical Protocol for Downstream Circulating-Free DNA Applications

This is a standard protocol for the processing and storage of blood samples for downstream liquid biopsy applications based on circulating free DNA. This is the first standard protocol published, although the technique has been in use for several years. It is easy to follow with standard equipment available in most translational or scientific laboratories.

The protocol can be performed by all laboratory staff. The protocol helps to standardize how we collect and store samples, which will ultimately impact in the clinic and reduce variability between different centers performing the same or similar downstream analysis where precision is of the outmost importance. This method is particularly useful in oncology research and also in clinical applications.

However, it may also be applied in other non-cancer diseases where the liquid biopsy is also used. After the blood sample has been centrifuged, knowing how much plasma or serum to remove can be difficult, but following this specific guidelines will help with this. The rest of the protocol is very easy to follow with limited experience in the laboratory.

Demonstrating the procedure will be Jorge, a technician from our laboratory. To begin, centrifuge the tube containing fresh blood at room temperature for 10 minutes at 1600 times g, with the maximum break applied. Carefully remove the tube from the centrifuge.

The upper phase of the serum supernatant will appear clear and yellowish. Check whether the sample shows signs of hemolysis and record the presence of hemolysis when appropriate. In a Class II biosafety cabinet, transfer the serum to collection tubes as 250-microliter aliquots.

The aliquots must be correctly labeled. Immediately freeze the serum upright in the storage box at minus 80 degrees Celsius, and record the time of sample storage. Verify that the samples were processed within the required four hours timeframe.

Centrifuge the EDTA tube at room temperature for 10 minutes at 1600 times g, with the maximum break applied. After centrifugation, the plasma supernatant will appear clear and yellowish. Check whether the sample shows signs of hemolysis and transfer the plasma to a 15-milliliter centrifuge tube without disturbing the cellular layer using a disposable serological pipette.

Leave a small residual volume of plasma above the cell layer at approximately five millimeters. If hemolysis is observed, discard the sample for further analysis. Centrifuge the plasma and a 15-milliliter centrifuge tube to remove any residual intact blood cells carried over from the first centrifugation step.

Carefully remove the tube from the centrifuge, not the cell pellet at the bottom of the tube, and transfer one to four milliliters of plasma to one to four milliliter polypropylene cryogenic bios or Eppendorfs using a disposable serological pipette. A residual volume of plasma must be left at the bottom of the tube to avoid contaminating the plasma with blood cells. Immediately freeze the plasma upright in the storage box at minus 80 degrees Celsius, and record the time of sample storage.

Verify that the samples are correctly labeled and were processed within the required four hours timeframe. Collect the cellular layer using a P1000 and filtered tip and transfer it to a two-milliliter tube. Immediately freeze and store at minus 80 degrees Celsius.

An example of a high quality cfDNA extraction that can be used for downstream applications is shown here, whereas this graphical image represents an example of an unsuitable sample with a high level of genomic contamination. The plasma or serum fraction must be a yellow color. This may vary depending on the individual sample.

Samples with hemolysis should be discarded. Be careful not to remove any cell pellet when removing the plasma fraction. This protocol can also be used for other nucleic acid based and protein applications, such as the detection of non-coding RNAs or proteins in serum and plasma samples using immuno detection techniques.

This is a very standard technique used in many laboratories. This protocol helps researchers to standardize how we perform our analysis as this is a crucial aspect, necessary for future clinical applications.