This manuscript was supported in part by the Korea Medical Device Development Fund (KMDF, Grant No. RS-2020-KD000019) and the Korea Health Industry Development Institute (KHIDI, Grant No. HI19C0521020020).

All whole blood samples were obtained from lung cancer patients. The research and analysis at Clinomics are carried out by the Cancer Genomics Research Institute, and IRB research approval by the government is led by the Asan Medical Center Institutional Review Committee (IRB NO. 2021-0802) with the IRB number registered for research at Clinomics.
1. Sample preparation
<ol>
	<li>Collect 9 mL of whole blood into an EDTA or cfDNA-stable blood collection tube.</li>
	<li>Mix well by flipping the tube up and down approximately 10 times.</li>
	<li>Store the samples at room temperature (RT; for short-term storage) or 4 &#176;C (for long-term storage). Do not freeze and thaw.</li>
</ol>
2. Device preparation
<ol>
	<li>Press the power switch to turn on the instrument. 
	NOTE: A loading screen appears on the touchpad, and the instrument is initialized. Keep hands away from the instrument during the initialization. The cartridge selection screen appears after the instrument initialization is completed.</li>
	<li>Select the sample mode to be used. Change the number of samples by pressing the arrow.</li>
</ol>
3. Device operation and sample collection
<ol>
	<li>Loading of the LBx-1 cartridge
	<ol>
		<li>Select the cartridge type on the touchscreen panel of the instrument. Change the number of samples by pressing the arrow button.</li>
		<li>Open the door of the instrument and insert all the cartridges to be used in order of the number on the cartridge holder. Make sure to correctly insert the cartridge and the cartridge holder. If the cartridge is not inserted correctly, it may cause considerable damage to the instrument.</li>
		<li>For a total of four cartridges, place the dummy cartridge in the empty space of the cartridge holder.</li>
		<li>Use the support wheel to mount the cartridge and tighten the lock nut to secure it.</li>
		<li>Close the door and press the RUN button on the touchpad screen. The instrument closes the valves on the cartridges, which takes approximately 30 s.</li>
		<li>Follow the message to open the door, remove the support wheel, and remove the valve-closed cartridge from the cartridge holder.</li>
		<li>Place the cartridge on the table and prepare to inject the whole blood sample. Pipette a maximum of 10 mL of whole blood sample using a serological pipette. 
		CAUTION: Dangers of handling blood. During the entire procedure of handling blood samples and reagents, it is important to wear a laboratory coat and laboratory gloves. The provided MSDS should be thoroughly studied by the laboratory workers.</li>
		<li>Insert the pipette tip deep into the sample inlet of the cartridge and slowly inject the whole blood sample. 
		NOTE When using more than or equal to 9 mL of whole blood sample, no phosphate buffered saline (PBS) addition is necessary. When using less than 9 mL of whole blood sample, add PBS to make the total volume to 9 mL. For example, when using 7.2 mL of whole blood, please add 1.8 mL of PBS into the sample inlet. A serological pipette or a pipette tip can be used for the injection of whole blood and PBS. When using less than 8 mL of whole blood sample, the buffy coat may not be recovered even by adding 1 mL of PBS. For gDNA prep, use 200 &#181;L of whole blood, which was separated before this step.</li>
		<li>Insert the cartridges to be used in order of the number on 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 OK button. 
		NOTE: Plasma and buffy coat are separated from whole blood automatically, which takes approximately 30 min. 
		CAUTION Danger during operation. Opening the door or touching the instrument during high-speed rotating operations can cause serious injuries. There is also a risk of injury due to asymmetric loading of the rotor. If unusual vibrations and noises occur when the instrument starts at assisted cell enrichment or expert mode, cartridge placement may be asymmetrical. Immediately press the power key to stop and properly install the cartridge.</li>
		<li>Look for the message on the screen accompanied by an alarm sound, which appears when the separation of plasma and buffy coat is complete.</li>
		<li>Stop the alarm by opening the door or pressing the STOP button. Open the door, remove the cartridge, and place it on the table.</li>
		<li>Recover 3 mL of plasma from the plasma outlet using a 1 mL pipette tip. Recover the buffy coat of 3 mL from the buffy coat outlet using a 1 mL pipette tip.</li>
	</ol>
	</li>
	<li>Loading the LBx-2 cartridge
	<ol>
		<li>Select the cartridge name on the touchscreen panel of the instrument. Change the number of samples by pressing the arrow button.</li>
		<li>Open the door and insert the cartridges to be used in order of the number on the cartridge holder.</li>
		<li>For a total of four cartridges, place the dummy cartridge in the empty space of the cartridge holder.</li>
		<li>Use the support wheel to mount the cartridge and tighten the lock nut to secure it. Ensure to correctly insert the cartridge in the cartridge holder. If the cartridge is not inserted correctly, it may cause considerable damage to the instrument.</li>
		<li>Close the door and press the RUN button on the touchpad screen. The instrument closes the valves on the cartridge, which takes approximately 30 s.</li>
		<li>Follow the message to open the door, remove the support wheel, and remove the valve-closed cartridge from the cartridge holder and place it on the table.</li>
		<li>Place the cartridge on the table and prepare to inject the density gradient solution and the whole blood sample. Check the volume of the density gradient solution and PBS to be injected depending on the volume of the whole blood sample (Supplementary Table 1).</li>
		<li>Pipette the density gradient solution using a serological pipette. Insert the pipette tip deep into the inlet of the cartridge and slowly inject the density gradient solution.</li>
		<li>After injecting the density gradient solution, pipette the 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. 
		NOTE: When using less than 9 mL of the whole blood sample, add PBS by referring to this table (Supplementary Table 1).</li>
		<li>Insert the cartridges to be used in order according to the number on 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 OK button.</li>
		<li>Plasma and PBMC are separated from whole blood automatically, which takes approximately 30 min. Look for the message that appears accompanied by an alarm sound, when the separation of plasma and PBMC is complete.</li>
		<li>Stop the alarm by opening the door or pressing the STOP button. Open the door, remove the cartridge, and place it on the table.</li>
		<li>Recover 3 mL of plasma from the plasma outlet using a 1 mL pipette tip. Recover 3 mL of the PBMC from the PBMC outlet using a 1 mL pipette tip.</li>
	</ol>
	</li>
	<li>Loading the FAST-auto cartridge
	<ol>
		<li>Select the cartridge on the touch screen panel of the instrument. Change the number of samples by pressing the arrow button.</li>
		<li>Open the door and insert 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.</li>
		<li>Use the support wheel to mount the cartridge and tighten the lock nut to secure it.</li>
		<li>Close the door and press the RUN button on the touch pad screen. The instrument closes the valves on the cartridge, which takes approximately 30 s.</li>
		<li>Follow the message to open the door, remove the support wheel, and remove the valve-closed cartridge from the cartridge holder.</li>
		<li>Place the cartridge on the table and prepare to inject the PBS solution and whole blood sample. Pipette 6 mL of PBS solution using a serological pipette. Insert the pipette tip deep into the PBS inlet of the cartridge and slowly inject 6 mL of the PBS solution.</li>
		<li>After injecting the PBS solution, pipette 3 mL of whole blood or PBMC sample obtained from LBx-2 using a serological pipette, which was previously rinsed with 1% BSA to prevent sticking of the residue. Insert the pipette tip deep into the sample inlet of the cartridge and slowly inject the whole blood sample.</li>
		<li>Insert the cartridges to be used in order of the number on 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 OK button.</li>
		<li>CTCs are enriched from whole blood automatically, which takes approximately 15 min. Look for a message that appears accompanied by an alarm sound when the enrichment of CTCs is complete. Stop the alarm by opening the door or pressing the STOP button.</li>
		<li>Open the door, remove the cartridge, and place it on the table. Insert the back plate remover (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.</li>
		<li>Carefully remove the cartridge body by lifting it up. Very gently pick up the filter membrane by the edge using a tweezer. Please make sure to use the outer rim (the 1 mm wide edge part) of the filter membrane to pinch and hold the filter membrane.</li>
		<li>Carefully place the filter membrane (on which enriched CTCs are residing) into a 1.5 mL tube for nucleic acid preparation. If necessary, recover the filtered blood to the blood outlet using a 1 mL pipette tip.</li>
	</ol>
	</li>
</ol>
4. Maintenance of the system
<ol>
	<li>Preparation for cleaning and disinfection of the instrument
	<ol>
		<li>Clean all the accessible surfaces of the instrument and the accessories once a week using ethanol and dried tissue, and also immediately when contaminated.</li>
		<li>Clean the bowl and rotor shaft regularly using alcohol (ethanol and isopropanol) or alcohol-based disinfectants.</li>
	</ol>
	</li>
	<li>Cleaning and disinfecting the instrument 
	NOTE: For general troubleshooting and other notes on the machine, refer to Supplementary Table 2.
	<ol>
		<li>Switch off the instrument using the main power switch. Disconnect the power plug from the power supply.</li>
		<li>Open the door and clean and disinfect all the accessible surfaces of the instrument, including the power cable, using a damp cloth and the recommended cleaning agents.</li>
		<li>Check the rotor shaft for damage. Inspect the instrument for corrosion and damage.</li>
		<li>Connect the instrument to the power supply only if it is fully dry inside and out.</li>
	</ol>
	</li>
	<li>Disconnect the main power plug and remove the fuse holder. The fuse holder is located above the power socket. Replace the used fuse with a spare one from the container.</li>
</ol>

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The authors have no conflicts of interest related to this work.

The amount and concentration of cfDNA and CTC depends on the individual, stage, and type of cancer. It also depends on the condition of the patient2,4,5,10,20. In particular, in the early or precancerous stages of cancer, the concentrations of cancer-related substances are very low, so there is a high possibility that it cannot be detected. Nevertheless, early detection has a very positive effect on patient survival and treatment strategy establishment. Since cfDNA and CTC have short half-life spans in the blood, they reflect real-time information about cancer21,22,23,24,25. Therefore, to simultaneously acquire various cancer-related substances, a large amount of blood is required under the same conditions. The system shown here provides the users with a simple method. The user can select and use the disc corresponding to the material required from whole blood. Using one of the given cartridges, plasma containing cfDNA can be obtained, and CTCs can be collected from the same blood sample, wasted buffy coat, or PBMC by re-injection into a FAST-auto disc (Figure 1).
Size-based CTC capture is simple, but it has limitations for purity. There are many different shapes and sizes of cells in the blood26. WBCs vary in size from 8 to 16 &#181;m27. In another study, the mean size of the measured WBC was 9 &#181;m. On the other hand, the minimum size of isolated CTCs is 16 &#181;m, and the average size is 30 &#181;m28. Furthermore, CTCs are present in a very small amount in the patient blood compared to other blood cells. Minimizing WBC contamination is important for downstream analyses. Although antibody-based detection can capture CTCs, high-resolution analysis such as NGS requires additional single-cell picking techniques or a high-level of bioinformatics analysis.
Recently, methods for intuitively observing the presence of CTCs before high-resolution analysis using various capture techniques has been implemented29. As shown in the results, the membrane acquired from the FAST-auto cartridge can be stained directly for CTC detection. Captured CTCs can also be easily resuspended from the membrane by vortexing for storage or further applications such as cell culture (Figure 1).
This method uses the principles of centrifugation and microfluidics. Therefore, the balance of the equipment and the disc is important, and it is also important to secure it with the disc holder. It is important to fill any initial insufficient total volume with PBS. If the initial amount is insufficient, there may be a problem leading to difficulties in moving to the next space. Take the disc holder out on a tabletop to prevent contamination of the equipment and to move liquids in and out of the disc. In the case of FAST-auto disc, process the samples quickly in order to reduce the damage to the CTC in the membrane. In particular, be careful to prevent contamination when handling the membrane and to follow the established protocol for long-term storage.
Nevertheless, this method is easy to use and can process one to four samples simultaneously. Also, various substrates of liquid can be obtained by simply replacing the disc. Moreover, no additional reagents are required other than using a density gradient solution to obtain PBMCs. Therefore, the obtained material is good for downstream use.
This method still has some limitations. The user cannot modify the disc arbitrarily, and different types of discs cannot be used at the same time. Also, there are limits to the maximum volume, so multiple discs must be used for large volumes of blood. Additional methods are still needed to obtain cfDNA. Using the membrane, it is difficult to obtain only CTCs using the pore-sized capture method. Therefore, cell selection or sorting is required for CTC-specific application experiments.
There are various fluids in the body, and the production of certain body fluids is associated with disease30. Currently, use of the blood body fluid has only been undertaken for analysis. In the future, an optimal protocol can be established by confirming the performance in various body fluids such as urine, ascites, pleural fluid, and spinal fluid. To provide a more convenient method for users, devices that automatically extract cfDNA from the disc are currently being developed. Furthermore, equipment capable of performing quantitative PCR directly within the disc following the extraction is under development.

Early and accurate detection of various diseases, including cancer, is the most important factor in establishing a treatment strategy1,2,3,4. In particular, early detection of cancer is closely related to increased survival chances for the patient5,6,7,8. Recently, liquid biopsies have been in the spotlight for the early detection of cancer. Solid tumors undergo angiogenesis and release various substances into the blood. In particular, circulating DNAs (ctDNAs), circulating RNAs (ctRNAs), proteins, vesicles such as exosomes, and circulating tumor cells (CTCs) have been found in the blood of cancer patients2,9. Although there are differences in the amount of these substances, they are consistently observed not only in the early stages but also in the later stages6,10. However, these individual differences are very high; for example, the amount of cell-free DNA (cfDNA) containing ctDNA is less than 1,000 ng, and the number of CTCs is less than 100 in 10 mL of whole blood from cancer patients11,12,13. Many studies have characterized cancer using these substances present in lesser amounts (i.e., cfDNA, ctDNA, and CTCs). To obtain accurate results, it is important to accurately separate small amounts of substances with high purity13,14. Conventional centrifugation methods are commonly used, but they are difficult to handle and have low purity depending on the user&#39;s skill. Since the discovery of CTCs, several separation techniques have been developed, such as centrifugation or density grade separation, immunobead, and microfluidic methods. Several containment techniques have been developed since the discovery of CTCs. However, these techniques are often limited when it is necessary to isolate cells from the various chips and membranes used to isolate them15. Also, the tagging methods require equipment such as FACS, and there are limits to the downstream process due to tagging contamination.
Recently, the use of liquid biopsies have increased, and various studies are being conducted for the early detection of cancer. Although this method is simple, there are still difficulties in downstream analysis, and various studies are attempting to overcome these difficulties16,17. In addition, many sites, including hospitals, require automated, reproducible, and high-purity methods that are convenient to use. Here, we have developed a lab-on-a-disc for the automated separation of substances from blood samples following a liquid biopsy. These devices are based on the principle of centrifugation, microfluidics, and pore-sized cell capture. There are three types of discs: LBx-1 can acquire plasma and buffy coat, while LBx-2 can acquire plasma and PBMC from whole blood with a volume of less than 10 mL; FAST-auto can also acquire CTCs using a membrane that is removable from the disc. Up to four of each disc can be used in one run. Above all, the advantage of this device and method is that it can obtain a variety of cancer-derived substances from the same sample using a small amount of blood. This means that the patient&#39;s blood only needs to be drawn once. In addition, it has the advantage of excluding errors due to differences in the blood sampling period. This platform is easy to use and provides accurate results for liquid biopsies and downstream applications. In this protocol, the usage of the device and cartridge is introduced.

<table><tbody><tr><td>1% BSA (Bovine Serum Albumin)</td><td>Sigma-Aldrich</td><td>A3059</td><td></td></tr><tr><td>1.5 mL Microcentrifuge Tube</td><td>Axygen</td><td>MCT-150-C-S</td><td></td></tr><tr><td>15 mL Conical Tube</td><td>SPL</td><td>50015</td><td></td></tr><tr><td>4150 TapeStation System</td><td>Agilent</td><td>G2992AA</td><td>Cell-free DNA Screen Tape (Agilent, 5067-5630), Cell-free DNA Sample Buffer (Agilent, 5067-5633)</td></tr><tr><td>Apostle MiniMax High Efficiency Cell-Free DNA Isolation Kit&amp;nbsp;</td><td>Apostle</td><td>A17622-250</td><td>5 mL X 50 preps version</td></tr><tr><td>BD Vacutainer blood collection tubes</td><td>BD</td><td>367525</td><td>EDTA Blood Collection Tube (10 mL)</td></tr><tr><td>BioViewCCBS</td><td>Clinomics</td><td></td><td>BioView Clinomics-Customized Bioview System. Allegro Plus microscope-based customization equipment</td></tr><tr><td>CD45 Monoclonal Antibody (HI30), PE-Alexa Fluor 610</td><td>Invitrogen</td><td>MHCD4522</td><td></td></tr><tr><td>FAST Auto cartridge</td><td>Clinomics</td><td>CLX-M3001</td><td></td></tr><tr><td>LBx-1 cartridge</td><td>Clinomics</td><td>CLX-M4101</td><td></td></tr><tr><td>LBx-2 cartridge</td><td>Clinomics</td><td>CLX-M4201</td><td></td></tr><tr><td>OPR-2000 instrument</td><td>Clinomics</td><td>CLX-I2001</td><td></td></tr><tr><td>Cover Glass</td><td>Marienfeld Superior</td><td>HSU-0101040</td><td></td></tr><tr><td>DynaMag 2 Magnet Stand</td><td>Thermo Fisher Scientific</td><td>12321D</td><td></td></tr><tr><td>Ficoll Paque Solution</td><td>GE healthcare</td><td>17-1440-03</td><td>density gradient solution</td></tr><tr><td>Filter Tip, 10 &amp;micro;L</td><td>Axygen</td><td>AX-TF-10</td><td>Pipette tips with aerosol barriers are recommended to help prevent cross contamination.</td></tr><tr><td>Filter Tip, 200 &amp;micro;L</td><td>Axygen</td><td>AX-TF-200</td><td>Pipette tips with aerosol barriers are recommended to help prevent cross contamination.</td></tr><tr><td>Filter Tip, 100 &amp;micro;L</td><td>Axygen</td><td>AX-TF-100</td><td>Pipette tips with aerosol barriers are recommended to help prevent cross contamination.</td></tr><tr><td>Filter Tip, 1000 &amp;micro;L</td><td>Axygen</td><td>AX-TF-1000</td><td>Pipette tips with aerosol barriers are recommended to help prevent cross contamination.</td></tr><tr><td>FITC anti-human CD326 (EpCAM) Antibody</td><td>BioLegend</td><td>324204</td><td></td></tr><tr><td>FITC Mouse Anti-Human Cytokeratin</td><td>BD Biosciences</td><td>347653</td><td></td></tr><tr><td>Formaldehyde solution (35 wt. % in H2O)</td><td>Sigma Aldrich</td><td>433284</td><td></td></tr><tr><td>Kimtech Science Wipers</td><td>Yuhan-Kimberly</td><td>41117</td><td></td></tr><tr><td>Latex glove</td><td>Microflex</td><td>63-754</td><td></td></tr><tr><td>Magnetic Bead Separation Rack</td><td>V&amp;amp;P Scientific</td><td>VP 772F2M-2</td><td></td></tr><tr><td>Manual Pipetting&amp;nbsp; (0.5-10 &amp;micro;L)</td><td>Eppendorf</td><td>3120000020</td><td></td></tr><tr><td>Manual Pipetting&amp;nbsp; (2-20 &amp;micro;L)</td><td>Eppendorf</td><td>3120000038</td><td></td></tr><tr><td>Manual Pipetting&amp;nbsp; (10-100 &amp;micro;L)</td><td>Eppendorf</td><td>3120000046</td><td></td></tr><tr><td>Manual Pipetting&amp;nbsp; (20-200 &amp;micro;L)</td><td>Eppendorf</td><td>3120000054</td><td></td></tr><tr><td>Manual Pipetting&amp;nbsp; (100-1000 &amp;micro;L)</td><td>Eppendorf</td><td>3120000062</td><td></td></tr><tr><td>Mounting Medium With DAPI&amp;nbsp;- Aqueous, Fluoroshield</td><td>abcam</td><td>ab104139</td><td></td></tr><tr><td>Normal Human IgG Control</td><td>R&amp;amp;D Systems</td><td>1-001-A</td><td></td></tr><tr><td>OLYMPUS BX-UCB</td><td>Olympus</td><td>9217316</td><td></td></tr><tr><td>Pan Cytokeratin Monoclonal Antibody (AE1/AE3), Alexa Fluor 488</td><td>Invitrogen</td><td>53-9003-82</td><td></td></tr><tr><td>PBS (Phosphate Buffered Saline Solution)</td><td>Corning</td><td>21-040CVC</td><td></td></tr><tr><td>Portable Pipet Aid</td><td>Drummond</td><td>4-000-201</td><td></td></tr><tr><td>Slide Glass</td><td>Marienfeld Superior</td><td>HSU-1000612</td><td></td></tr><tr><td>StainTray Staining box</td><td>Simport</td><td>M920</td><td></td></tr><tr><td>Sterile Serological Pipette (10 mL)</td><td>SPL</td><td>91010</td><td></td></tr><tr><td>Triton&amp;nbsp;X-100 solution</td><td>Sigma Aldrich</td><td>93443</td><td></td></tr><tr><td>TWEEN&amp;nbsp;20</td><td>Sigma Aldrich</td><td>P7949</td><td></td></tr><tr><td>Whole Blood</td><td></td><td></td><td>Stored at 4-8 &amp;deg;C by collecting in EDTA or cfDNA stable tube : If the whole blood is insufficient in 9 mL, add PBS (phosphate buffered saline) as much as necessary.</td></tr><tr><td>X-Cite 120Q (Fluorescence Lamp Illuminator)</td><td>Excelitas</td><td>010-00157</td><td></td></tr></tbody></table>

automatic separation and collection of cancer-related substances from clinical samples

Recently, liquid biopsies have been used to diagnose various diseases, including cancer. Body fluids contain many substances, including cells, proteins, and nucleic acids originating from normal tissues, but some of these substances also originate from the diseased area. The investigation and analysis of these substances in the body fluids play a pivotal role in the diagnosis of various diseases. Therefore, it is important to accurately separate the required substances, and several techniques are developed to be used for this purpose.
We have developed a lab-on-a-disc type of device and platform named CD-PRIME. This device is automated and has good results for sample contamination and sample stability. Moreover, it has advantages of a good acquisition yield, a short operation time, and high reproducibility. In addition, depending on the type of disc to be mounted, plasma containing cell-free DNA, circulating tumor cells, peripheral blood mononuclear cells, or buffy coats can be separated. Thus, the acquisition of a variety of materials present in the body fluids can be done for a variety of downstream applications, including the study of omics.

The goal of this technique is to easily and automatically isolate cancer-associated substances from whole blood. In particular, anyone can use this technique in all the suitable fields of research and analysis. The simultaneous and reproducible separation of multiple substances in a single blood sample is significant in liquid biopsies. The LBx-1 and LBx-2 discs are used for isolating plasma and buffy coat or PBMC from whole blood. Figure 1 shows the materials separated by the application of this device. First, the plasma was obtained from 10 mL of blood using LBx-1 or the PBMCs were obtained using LBx-2. Second, 3 mL of the separated buffy coat was re-injected with PBS into FAST-auto, and CTCs were filtered through the membrane. Third, the membrane was transferred to a tube filled with storage buffer or immediately used for staining. For other applications or long-term storage, CTCs can be easily removed from the membrane by vortexing.
cfDNA was extracted from the plasma using magnetic bead-based DNA isolation capable of size-by-size capture of DNA by bead concentration. The concentration and purity of cfDNA were measured using the Bioanalyzer. It is well known that the concentration of cfDNA, including ctDNA, varies depending on the type and stage of cancer18. In addition, most cfDNA has a length of 166 bp, and some are about twice or thrice as long. Although there is a difference in the amount obtained from each individual sample, such as 8.47 ng/mL and 5.2 ng/mL, the results of cfDNA extraction were good for both concentration and size distribution in all cases (Figure 2). 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 for the detection of CTCs and white blood cells (WBCs). The stained membrane placed on glass slides were observed under a fluorescence microscope. Staining and microscopic studies were carried out following a previous study19. Only CTCs can be specifically distinguished using the EpCAM/Cytokeratin (CTC positive marker) and CD45 (WBC positive marker) antibodies. Each membrane captured a total of 310 and 998 cells, respectively. Among them, a total of 3 and 43 CTCs were counted in samples 1 and 2, respectively (Figure 3). The method used in this study makes it easy to determine the presence and number of CTCs. In addition, changes in the cfDNA concentration and the CTC number can be used to easily monitor the presence and recurrence of cancer in the field. In particular, since these methods do not use other reagents that may affect the results, downstream experiments are possible with the obtained material.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/64325/64325fig01.jpg" /> 
Figure 1: Workflow of disc mixing method for simultaneous separation of substances from whole blood. (A) The plasma containing cfDNA is obtained. (B) CTCs are removed from the buffy coat. (C,D) After vortex, CTCs are detached from the membrane, and a small amount of CTC still remains on the membrane. Both obtained cells and the membrane can undergo long-term storage by freezing samples in the storage buffer. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/64325/64325fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/64325/64325fig02.jpg" /> 
Figure 2: Purity and amount of extracted cfDNA. The extracted cfDNAs were evaluated using Bioanalyzer, and cfDNA screen tapes were used together with an electronic ladder. The green line was used for alignment between the ladder (left) and the sample (right). The triangle marks indicate the DNA band. Most of cfDNA is 166 bp, and some exist in integer multiples of length. Usually, the concentration of cfDNA measures up to the size of 50 bp to 700 bp. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/64325/64325fig02large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/64325/64325fig03.jpg" /> 
Figure 3: Counting of CTCs using membrane staining. After membrane immunocytochemistry staining, cells show DAPI (Nuclear positive) signals in blue, while EpCAM/CK (CTCs positive) and CD45 (WBC positive) are highlighted in green and red, respectively. Fluorescence signals were measured using a fluorescence microscope. The scale bar indicates 10 &#181;m. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/64325/64325fig03large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
Supplementary Table 1: Composition of the final volume for density gradient solution use. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/64325/Supplementary Table 1_Revision_Bae et al_RE.xlsx" target="_blank">Please click here to download this File.</a>
Supplementary Table 2: General issues and cautions for maintenance. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/64325/Supplementary Table 2_Revision_Bae et al_RE.xlsx" target="_blank">Please click here to download this File.</a>

Watch this Scientific Journal Video about Automatic Separation and Collection of Cancer-Related Substances from Clinical Samples at JoVE.com

Automatic Separation and Collection of Cancer-Related Substances from Clinical Samples

This paper describes the application of automated equipment to easily and efficiently separate and collect substances, such as cell-free DNA and circulating tumor cells, from whole blood.

Recently, liquid biopsies have been used to diagnose various diseases, including cancer. Body fluids contain many substances, including cells, proteins, ...

automatic-separation-collection-cancer-related-substances-from

Nanyang Technological University

Research

JoVE Journal

Cancer Research

1.8K Views.  Clinomics Inc.. This paper describes the application of automated equipment to easily and efficiently separate and collect substances, such as cell-free DNA and circulating tumor cells, from whole blood. 

Video: Automatic Separation and Collection of Cancer-Related Substances from Clinical Samples

Generation of Comprehensive Thoracic Oncology Database - Tool for Translational Research

The Thoracic Oncology Program Database Project was created to serve as a comprehensive, verified, and accessible repository for well-annotated cancer specimens and clinical data to be available to researchers within the Thoracic Oncology Research Program. This database also captures a large volume of genomic and proteomic data obtained from various tumor tissue studies. A team of clinical and basic science researchers, a biostatistician, and a bioinformatics expert was convened to design the database. Variables of interest were clearly defined and their descriptions were written within a standard operating manual to ensure consistency of data annotation. Using a protocol for prospective tissue banking and another protocol for retrospective banking, tumor and normal tissue samples from patients consented to these protocols were collected. Clinical information such as demographics, cancer characterization, and treatment plans for these patients were abstracted and entered into an Access database. Proteomic and genomic data have been included in the database and have been linked to clinical information for patients described within the database. The data from each table were linked using the relationships function in Microsoft Access to allow the database manager to connect clinical and laboratory information during a query. The queried data can then be exported for statistical analysis and hypothesis generation.

A thoracic oncology database was developed to serve as a comprehensive repository for clinical and laboratory data for the purposes of translational research. The database will serve translational cancer researchers within the Thoracic Oncology Research Program. This database is adaptable to other cancer models, as well as other human diseases.

The Thoracic Oncology Program Database Project was created to serve as a comprehensive, verified, and accessible repository for well-annotated cancer ...

Medicine

Rapid Isolation of Viable Circulating Tumor Cells from Patient Blood Samples

Circulating tumor cells (CTC) are cells that disseminate from a primary tumor throughout the circulatory system and that can ultimately form secondary tumors at distant sites. CTC count can be used to follow disease progression based on the correlation between CTC concentration in blood and disease severity1. As a treatment tool, CTC could be studied in the laboratory to develop personalized therapies. To this end, CTC isolation must cause no cellular damage, and contamination by other cell types, particularly leukocytes, must be avoided as much as possible2. Many of the current techniques, including the sole FDA-approved device for CTC enumeration, destroy CTC as part of the isolation process (for more information see Ref. 2). A microfluidic device to capture viable CTC is described, consisting of a surface functionalized with E-selectin glycoprotein in addition to antibodies against epithelial markers3. To enhance device performance a nanoparticle coating was applied consisting of halloysite nanotubes, an aluminosilicate nanoparticle harvested from clay4. The E-selectin molecules provide a means to capture fast moving CTC that are pumped through the device, lending an advantage over alternative microfluidic devices wherein longer processing times are necessary to provide target cells with sufficient time to interact with a surface. The antibodies to epithelial targets provide CTC-specificity to the device, as well as provide a readily adjustable parameter to tune isolation. Finally, the halloysite nanotube coating allows significantly enhanced isolation compared to other techniques by helping to capture fast moving cells, providing increased surface area for protein adsorption, and repelling contaminating leukocytes3,4. This device is produced by a straightforward technique using off-the-shelf materials, and has been successfully used to capture cancer cells from the blood of metastatic cancer patients. Captured cells are maintained for up to 15 days in culture following isolation, and these samples typically consist of &gt;50% viable primary cancer cells from each patient. This device has been used to capture viable CTC from both diluted whole blood and buffy coat samples. Ultimately, we present a technique with functionality in a clinical setting to develop personalized cancer therapies.

Circulating tumor cells are isolated from the blood of cancer patients without inflicting cellular damage. Isolation of tumor cells is accomplished using a bimolecular surface of E-selectin in addition to antibodies against epithelial markers. A nanotube coating specifically promotes cancer cell adhesion resulting in high capture purities.

Circulating tumor cells (CTC) are cells that disseminate from a primary tumor throughout the circulatory system and that can ultimately form secondary ...

Bioengineering

Detection and Monitoring of Tumor Associated Circulating DNA in Patient Biofluids

Complications associated with upfront and repeat surgical tissue sampling present the need for minimally invasive platforms capable of molecular sub-classification and temporal monitoring of tumor response to therapy. Here, we describe our dPCR-based method for the detection of tumor somatic mutations in cell free DNA (cfDNA), readily available in&#160;patient biofluids. Although limited in the number of mutations that can be tested for in each assay, this method provides a high level of sensitivity and specificity. Monitoring of mutation abundance, as calculated by MAF, allows for the evaluation of tumor response to therapy, thereby providing a much-needed supplement to radiographic imaging.

Here, we present a protocol to detect tumor somatic mutations in circulating DNA present in patient biological fluids (biofluids). Our droplet digital polymerase chain reaction (dPCR)-based method enables quantification of the tumor mutation allelic frequency (MAF), facilitating a minimally invasive complement to diagnosis and temporal monitoring of tumor response.

Complications associated with upfront and repeat surgical tissue sampling present the need for minimally invasive platforms capable of molecular ...

Semi-automatic PD-L1 Characterization and Enumeration of Circulating Tumor Cells from Non-small Cell Lung Cancer Patients by Immunofluorescence

Circulating tumor cells (CTCs) derived from the primary tumor are shed into the bloodstream or lymphatic system. These rare cells (1&#8722;10 cells per mL of blood) warrant a poor prognosis and are correlated with shorter overall survival in several cancers (e.g., breast, prostate and colorectal). Currently, the anti-EpCAM-coated magnetic bead-based CTC capturing system is the gold standard test approved by the U.S. Food and Drug Administration (FDA) for enumerating CTCs in the bloodstream. This test is based on the use of magnetic beads coated with anti-EpCAM markers, which specifically target epithelial cancer cells. Many studies have illustrated that EpCAM is not the optimal marker for CTC detection. Indeed, CTCs are a heterogeneous subpopulation of cancer cells and are able to undergo an epithelial-to-mesenchymal transition (EMT) associated with metastatic proliferation and invasion. These CTCs are able to reduce the expression of cell surface epithelial marker EpCAM, while increasing mesenchymal markers such as vimentin. To address this technical hurdle, other isolation methods based on physical properties of CTCs have been developed. Microfluidic technologies enable a label-free approach to CTC enrichment from whole blood samples. The spiral microfluidic technology uses the inertial and Dean drag forces with continuous flow in curved channels generated within a spiral microfluidic chip. The cells are separated based on the differences in size and plasticity between normal blood cells and tumoral cells. This protocol details the different steps to characterize the programmed death-ligand 1 (PD-L1) expression of CTCs, combining a spiral microfluidic device with customizable immunofluorescence (IF) marker set.

The characterization of circulating tumor cells (CTCs) is a popular topic in translational research. This protocol describes a semi-automatic immunofluorescence (IF) assay for PD-L1 characterization and enumeration of CTCs in non-small cell lung cancer (NSCLC) patient samples.

Circulating tumor cells (CTCs) derived from the primary tumor are shed into the bloodstream or lymphatic system. These rare cells (1&#8722;10 cells per mL ...

Detection of Cell-Free DNA in Blood Plasma Samples of Cancer Patients

Identifying mutations in tumors of cancer patients is a very important step in disease management. These mutations serve as biomarkers for tumor diagnosis as well as for the treatment selection and its response in cancer patients. The current gold standard method for detecting tumor mutations involves a genetic test of tumor DNA by means of tumor biopsies. However, this invasive method is difficult to be performed repeatedly as a follow-up test of the tumor mutational repertoire. Liquid biopsy is a new and emerging technique for detecting tumor mutations as an easy-to-use and non-invasive biopsy approach.
Cancer cells multiply rapidly. In parallel, numerous cancer cells undergo apoptosis. Debris from these cells are released into a patient&#8217;s circulatory system, together with finely fragmented DNA pieces, called cell-free DNA (cfDNA) fragments, which carry tumor DNA mutations. Therefore, for identifying cfDNA based biomarkers using liquid biopsy technique, blood samples are collected from the cancer patients, followed by the separation of plasma and buffy coat. Next, plasma is processed for the isolation of cfDNA, and the respective buffy coat is processed for the isolation of a patient&#39;s genomic DNA. Both nucleic acid samples are then checked for their quantity and quality; and analyzed for mutations using next-generation sequencing (NGS) techniques.
In this manuscript, we present a detailed protocol for liquid biopsy, including blood collection, plasma, and buffy coat separation, cfDNA and germline DNA extraction, quantification of cfDNA or germline DNA, and cfDNA fragment enrichment analysis.

In this paper we present a detailed protocol for non-invasive liquid biopsy technique, including blood collection, plasma and buffy coat separation, cfDNA and germline DNA extraction, quantification of cfDNA or germline DNA, and cfDNA fragment enrichment analysis.

Identifying mutations in tumors of cancer patients is a very important step in disease management. These mutations serve as biomarkers for tumor diagnosis ...

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

The term liquid biopsy (LB) refers to molecules such as proteins, DNA, RNA, cells, or extracellular vesicles in blood and other bodily fluids that originate from the primary and/or metastatic tumor. LB has emerged as a mainstay in translational research and has started to become part of clinical oncology practice, providing a minimally invasive alternative to solid biopsy. The LB allows real-time monitoring of a tumor via a minimally invasive sample extraction, such as blood. The applications include early cancer detection, patient follow-up for the detection of disease progression, assessment of minimal residual disease, and potential identification of molecular progression and mechanism of resistance. In order to achieve a reliable analysis of these samples that can be reported in the clinic, the preanalytical procedures should be carefully considered and strictly followed. Sample collection, quality, and storage are crucial steps that determine their usefulness in downstream applications. Here, we present standardized protocols from our liquid biopsy working module for collecting, processing, and storing plasma and serum samples for downstream liquid biopsy analysis based on circulating-free DNA. The protocols presented here require standard equipment and are sufficiently flexible to be applied in most laboratories focused on biological procedures.

The liquid biopsy has revolutionized our approach to oncology translational studies, with sample collection, quality, and storage being crucial steps for its successful clinical application. Here we describe a standardized and validated protocol for downstream circulating-free DNA applications that can be applied in most translational research laboratories.

The term liquid biopsy (LB) refers to molecules such as proteins, DNA, RNA, cells, or extracellular vesicles in blood and other bodily fluids that ...

Adaptation of Semiautomated Circulating Tumor Cell (CTC) Assays for Clinical and Preclinical Research Applications

The majority of cancer-related deaths occur subsequent to the development of metastatic disease. This highly lethal disease stage is associated with the presence of circulating tumor cells (CTCs). These rare cells have been demonstrated to be of clinical significance in metastatic breast, prostate, and colorectal cancers. The current gold standard in clinical CTC detection and enumeration is the FDA-cleared CellSearch system (CSS). This manuscript outlines the standard protocol utilized by this platform as well as two&#160;additional adapted protocols that describe the detailed process of user-defined marker optimization for protein characterization of patient CTCs and a comparable protocol for CTC capture in very low volumes of blood, using standard CSS reagents, for studying in vivo preclinical mouse models of metastasis. In addition, differences in CTC quality between healthy donor blood spiked with cells from tissue culture versus patient blood samples are highlighted. Finally, several commonly discrepant items that can lead to CTC misclassification errors are outlined. Taken together, these protocols will provide a useful resource for users of this platform interested in preclinical and clinical research pertaining to metastasis and CTCs.

Adaptation of Semiautomated Circulating Tumor Cell CTC Assays for Clinical and Preclinical Research Applications

Circulating tumor cells (CTCs) are prognostic in several metastatic cancers. This manuscript describes the gold standard CellSearch system (CSS) CTC enumeration platform and highlights common misclassification errors. In addition, two&#160;adapted protocols are described for user-defined marker characterization of CTCs and CTC enumeration in preclinical mouse models of metastasis using this technology.

The majority of cancer-related deaths occur subsequent to the development of metastatic disease. This highly lethal disease stage is associated with the ...

Quantitative Mass Spectrometric Profiling of Cancer-cell Proteomes Derived From Liquid and Solid Tumors

In-depth analyses of cancer cell proteomes are needed to elucidate oncogenic pathomechanisms, as well as to identify potential drug targets and diagnostic biomarkers. However, methods for quantitative proteomic characterization of patient-derived tumors and in particular their cellular subpopulations are largely lacking. Here we describe an experimental set-up that allows quantitative analysis of proteomes of cancer cell subpopulations derived from either liquid or solid tumors. This is achieved by combining cellular enrichment strategies with quantitative Super-SILAC-based mass spectrometry followed by bioinformatic data analysis. To enrich specific cellular subsets, liquid tumors are first immunophenotyped by flow cytometry followed by FACS-sorting; for solid tumors, laser-capture microdissection is used to purify specific cellular subpopulations. In a second step, proteins are extracted from the purified cells and subsequently combined with a tumor-specific, SILAC-labeled spike-in standard that enables protein quantification. The resulting protein mixture is subjected to either gel electrophoresis or Filter Aided Sample Preparation (FASP) followed by tryptic digestion. Finally, tryptic peptides are analyzed using a hybrid quadrupole-orbitrap mass spectrometer, and the data obtained are processed with bioinformatic software suites including MaxQuant. By means of the workflow presented here, up to 8,000 proteins can be identified and quantified in patient-derived samples, and the resulting protein expression profiles can be compared among patients to identify diagnostic proteomic signatures or potential drug targets.

In-depth analyses of cancer cell proteomes facilitate identification of novel drug targets and diagnostic biomarkers. We describe an experimental workflow for quantitative analysis of (phospho-)proteomes in cancer cell subpopulations derived from liquid and solid tumors. This is achieved by combining cellular enrichment strategies with quantitative Super-SILAC-based mass spectrometry.

In-depth analyses of cancer cell proteomes are needed to elucidate oncogenic pathomechanisms, as well as to identify potential drug targets and diagnostic ...

Establishment of a Clinic-based Biorepository

The incidence of skin cancer (e.g., squamous cell carcinoma, basal cell carcinoma, and melanoma) has been increasing over the past several years. It is expected that there will be a parallel demand for cutaneous tumor samples for biomedical research studies. Tissue availability, however, is limited due the cost of establishing a biorepository and the lack of protocols available for obtaining clinical samples that do not interfere with clinical operations. A protocol was established to collect and process cutaneous tumor and associated blood and saliva samples that has minimal impact on routine clinical procedures on the date of a Mohs surgery. Tumor samples are collected and processed from patients undergoing their first layer of Mohs surgery for biopsy-proven cutaneous malignancies by the Mohs histotechnologist. Adjacent normal tissue is collected at the time of surgical closure. Additional samples that may be collected are whole-blood and buccal swabs. By utilizing tissue samples that are normally discarded, a biorepository was generated that offers several key advantages by being based in the clinic versus the laboratory setting. These include a wide range of collected samples; access to de-identified patient records, including pathology reports; and, for the typical donor, access to additional samples during follow-up visits.

Cutaneous tumors are often discarded following Mohs micrographic surgery. A protocol is described here that enables clinical support staff to effectively process and store cutaneous tumor (e.g., squamous cell carcinoma, basal cell carcinoma, and melanoma) samples for downstream laboratory applications without interfering with clinical operations.

The incidence of skin cancer (e.g., squamous cell carcinoma, basal cell carcinoma, and melanoma) has been increasing over the past several years. It is ...

Clinical Microfluidic Chip Platform for the Isolation of Versatile Circulating Tumor Cells

Circulating tumor cells (CTCs) are significant in cancer prognosis, diagnosis, and anti-cancer therapy. CTC enumeration is vital in determining patient disease since CTCs are rare and heterogeneous. CTCs are detached from the primary tumor, enter the blood circulation system, and potentially grow at distant sites, thus metastasizing the tumor. Since CTCs carry similar information to the primary tumor, CTC isolation and subsequent characterization can be critical in monitoring and diagnosing cancer. The enumeration, affinity modification, and clinical immunofluorescence staining of rare CTCs are powerful methods for CTC isolation because they provide the necessary elements with high sensitivity. Microfluidic chips offer a liquid biopsy method that is free of any pain for the patients. In this work, we present a list of protocols for clinical microfluidic chips, a versatile CTC isolating platform, that incorporate a set of functionalities and services required for CTC separation, analysis, and early diagnosis, thus facilitating biomolecular analysis and cancer treatment. The program includes rare tumor cell counting, clinical patient blood preprocessing, which includes red blood cell lysis, and the isolation and recognition of CTCs in situ on microfluidic chips. The program allows the precise enumeration of tumor cells or CTCs. Additionally, the program includes a tool that incorporates CTC isolation with versatile microfluidic chips and immunofluorescence identification in situ on the chips, followed by biomolecular analysis.

The clinical microfluidic chip is an important biomedical analysis technique that simplifies clinical patient blood sample preprocessing and immunofluorescently stains circulating tumor cells (CTCs) in situ on the chip, allowing the rapid detection and identification of a single CTC.

Circulating tumor cells (CTCs) are significant in cancer prognosis, diagnosis, and anti-cancer therapy. CTC enumeration is vital in determining patient ...