Our protocol is user-friendly. Using this technique, anyone can easily isolated component from the blood. Contamination can be minimized and materials can be separated accurately.
With automation and the establishment of this protocol, researchers can easily obtain cancer-related substances with high purity, which is helpful for various downstream studies. In particular, it is expected to provide important advantages for end user research. Demonstrating their procedure will be Jay Jeong, a senior researcher and assistant manager at Cancer Genomics Research Institute.
To begin, load the LBx 1 cartridge by selecting the cartridge type on the touchscreen panel of the instrument. Press the arrow button to change the number of samples. Then, open the instrument door and insert all the cartridges to be used in order of the number on the cartridge holder.
For a total of four cartridges, place the dummy cartridge in the empty space of the cartridge holder. Use the support wheel to mount the cartridge and tighten the lock nut to secure it. Close the door, and press the run button on the touchpad screen.
The instrument closes the valves on the cartridges which takes approximately 30 seconds. Follow the message to open the door. Remove the support wheel and remove the valve-closed cartridge from the cartridge holder.
Pipette a maximum of 10 milliliters of a whole blood sample using a serological pipette. Insert the pipette tip deep into the sample inlet of the cartridge, and slowly inject the whole blood sample. After inserting the cartridges into the instrument, close the door of the instrument and press the OK button.
Plasma and buffy coats are automatically separated from whole blood, which takes approximately 30 minutes. Once the plasma and buffy coat are separated, a message on the screen accompanied by an alarm sound will appear. Stop the alarm by opening the door or pressing the stop button.
Open the door. Remove the cartridge and place it on the table. Recover three milliliters of plasma from the plasma outlet using a one-milliliter pipette tip.
Then, recover three milliliters of the buffy coat from the buffy coat outlet using a one-milliliter pipette tip. Next, load the LBx 2 cartridge by selecting the cartridge name on the touchscreen panel of the instrument. Press the arrow button to change the number of samples and follow the same procedure shown for the LBx 1 cartridge to insert the cartridge in the cartridge holder.
After inserting the cartridge and closing the door, press the run button on the touchpad screen. Follow the procedure shown previously to remove the valve-closed cartridge from the cartridge holder and place it on the table. Pipette the density gradient solution using a serological pipette.
Insert the pipette tip deep into the inlet of the cartridge and slowly inject the solution. Then, pipette the whole blood sample and slowly inject it into the sample inlet. After inserting and securing the cartridge, close the door and press the OK button.
Once the plasma and PBMC are automatically separated from whole blood, a message accompanied by an alarm sound will appear. Open the door and remove the cartridge. Recover three milliliters of plasma from the plasma outlet using a one-milliliter pipette tip, and then, recover three milliliters of the PBMC from the PBMC outlet.
Similarly, for the FAST-auto cartridge, after removing the valve-closed cartridge from the cartridge holder, inject six milliliters of the PBS solution into the PBS inlet of the cartridge. Then, pipette three milliliters of whole blood or buffy coat obtained from LBx 1, or PBMC sample obtained from LBx 2, and slowly inject the sample into the sample inlet of the cartridge. After inserting the cartridge and securing it, close the door and press the OK button.
Once the automatic enrichment of CTCs is complete, stop the alarm. Remove the cartridge and insert the backplate remover, or BPR, into the four holes on the front of the cartridge. Press the dark blue wing with the thumbs of both hands and then, press the light blue body until it clicks.
Carefully remove the cartridge body by lifting it. Very gently, pick up the filter membrane by the edge using a tweezer, and place the membrane into a 1.5-milliliter tube for nucleic acid preparation, or on a slide glass for the staining. If necessary, recover the filtered blood to the blood outlet using a one-milliliter pipette tip.
The extracted cfDNAs were evaluated using Bioanalyzer, and cfDNA screen tapes were used together with an electronic ladder. The green line was used to align the ladder and the sample. The triangle marks indicate the DNA band.
Most cfDNA has a length of 166 base pairs and some exist in integer multiples of length. Usually, the concentration of cfDNA measures up to the size of 50 to 700 base pairs. Although there is a difference in the amount obtained from each individual sample, such as 8.47 nanograms per milliliter, and 5.2 nanograms per milliliter, the results of cfDNA extraction were good for both concentration and size distribution in all cases.
These results indicate that the LBx 1 method accurately separates plasma containing cfDNA. In FAST-auto, the membrane was removed and stained with fluorescent antibodies to detect CTCs and white blood cells. The stained membrane, placed on glass slides, was observed under a fluorescence microscope.
After membrane staining, cells show DAPI or nuclear-positive signals in blue, while EpCAM and CK or CTCs positive are highlighted in green, and CD45 or WBC positive are highlighted in red. It is not difficult to remove the membrane. However, it is necessary to be careful about the loss and contamination of CTCs.
Whole blood, PBMC, and buffy coat are all available. Instead, you just need to set the entire volume. Other liquids may also be used, although testing will be required.
This technique can be operated directly in hospitals and the substance can be obtained quickly.
