Name: automatic separation and collection of cancer-related substances from clinical samples
Uploaded: 2023-01-13T14:40:00
Description: Watch this Scientific Journal Video about Automatic Separation and Collection of Cancer-Related Substances from Clinical Samples at JoVE.com

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 wel...

<ol>
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W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Centrifugation+affects+the+purity+of+liquid+biopsy-based+tumor+biomarkers.">Centrifugation affects the purity of liquid biopsy-based tumor biomarkers.</a> Cytometry Part A. 93 (12), 1207-1212 (2018).</li><li>Sharma, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Circulating+tumor+cell+isolation,+culture,+and+downstream+molecular+analysis.">Circulating tumor cell isolation, culture, and downstream molecular analysis.</a> Biotechnology advances. 36 (4), 1063-1078 (2018).</li><li>Bennett, C. W., Berchem, G., Kim, Y. J., El-Khoury, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell-free+DNA+and+next-generation+sequencing+in+the+service+of+personalized+medicine+for+lung+cancer.">Cell-free DNA and next-generation sequencing in the service of personalized medicine for lung cancer.</a> Oncotarget. 7 (43), 71013 (2016).</li><li>Lowes, L. E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Circulating+tumor+cells+(CTC)+and+cell-free+DNA+(cfDNA)+workshop+2016:+scientific+opportunities+and+logistics+for+cancer+clinical+trial+incorporation.">Circulating tumor cells (CTC) and cell-free DNA (cfDNA) workshop 2016: scientific opportunities and logistics for cancer clinical trial incorporation.</a> International Journal of Molecular Sciences. 17 (9), 1505 (2016).</li><li>Bryzgunova, O. E., Konoshenko, M. Y., Laktionov, P. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Concentration+of+cell-free+DNA+in+different+tumor+types.">Concentration of cell-free DNA in different tumor types.</a> Expert Review of Molecular Diagnostics. 21 (1), 63-75 (2021).</li><li>Park, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Circulating+tumour+cells+as+an+indicator+of+early+and+systemic+recurrence+after+surgical+resection+in+pancreatic+ductal+adenocarcinoma.">Circulating tumour cells as an indicator of early and systemic recurrence after surgical resection in pancreatic ductal adenocarcinoma.</a> Scientific Reports. 11 (1), 1-12 (2021).</li><li>Heidrich, I., A&#269;kar, L., Mossahebi Mohammadi, P., Pantel, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Liquid+biopsies:+Potential+and+challenges.">Liquid biopsies: Potential and challenges.</a> International Journal of Cancer. 148 (3), 528-545 (2021).</li><li>Celec, P., Vlkov&#225;, B., Laukov&#225;, L., B&#225;b&#237;&#269;kov&#225;, J., Boor, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell-free+DNA:+the+role+in+pathophysiology+and+as+a+biomarker+in+kidney+diseases.">Cell-free DNA: the role in pathophysiology and as a biomarker in kidney diseases.</a> Expert Reviews in Molecular Medicine. 20, 1 (2018).</li><li>Thierry, A. R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Origin+and+quantification+of+circulating+DNA+in+mice+with+human+colorectal+cancer+xenografts.">Origin and quantification of circulating DNA in mice with human colorectal cancer xenografts.</a> Nucleic Acids Research. 38 (18), 6159-6175 (2010).</li><li>Moreira, V. G., de la Cera Mart&#237;nez, T., Gonzalez, E. G., Garcia, B. P., Menendez, F. V. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Increase+in+and+clearance+of+cell-free+plasma+DNA+in+hemodialysis+quantified+by+real-time+PCR.">Increase in and clearance of cell-free plasma DNA in hemodialysis quantified by real-time PCR.</a> Clinical Chemistry and Laboratory Medicine (CCLM). 44 (12), 1410-1415 (2006).</li><li>Gauthier, V. J., Tyler, L. N., Mannik, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Blood+clearance+kinetics+and+liver+uptake+of+mononucleosomes+in+mice.">Blood clearance kinetics and liver uptake of mononucleosomes in mice.</a> Journal of Immunology. 156 (3), 1151-1156 (1996).</li><li>Meng, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Circulating+tumor+cells+in+patients+with+breast+cancer+dormancy.">Circulating tumor cells in patients with breast cancer dormancy.</a> Clinical Cancer Research. 10 (24), 8152-8162 (2004).</li><li>Alix-Panabi&#232;res, C., Pantel, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Challenges+in+circulating+tumour+cell+research.">Challenges in circulating tumour cell research.</a> Nature Reviews Cancer. 14 (9), 623-631 (2014).</li><li>Zhou, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+of+circulating+tumor+cells+in+non-small-cell-lung-cancer+patients+using+a+multi-flow+microfluidic+channel.">Isolation of circulating tumor cells in non-small-cell-lung-cancer patients using a multi-flow microfluidic channel.</a> Microsystems &amp; Nanoengineering. 5 (1), 8 (2019).</li><li>Sajay, B. N. G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Towards+an+optimal+and+unbiased+approach+for+tumor+cell+isolation.">Towards an optimal and unbiased approach for tumor cell isolation.</a> Biomedical Microdevices. 15 (4), 699-709 (2013).</li><li>Bailey, P. C., Martin, S. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Insights+on+CTC+biology+and+clinical+impact+emerging+from+advances+in+capture+technology.">Insights on CTC biology and clinical impact emerging from advances in capture technology.</a> Cells. 8 (6), 553 (2019).</li><li>Ahn, S. M., Simpson, R. 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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...

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 border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<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&#160;</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 &#181;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 &#181;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 &#181;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 &#181;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&#38;P Scientific</td>
			<td>VP 772F2M-2</td>
			<td></td>
		</tr>
		<tr>
			<td>Manual Pipetting&#160; (0.5-10 &#181;L)</td>
			<td>Eppendorf</td>
			<td>3120000020</td>
			<td></td>
		</tr>
		<tr>
			<td>Manual Pipetting&#160; (2-20 &#181;L)</td>
			<td>Eppendorf</td>
			<td>3120000038</td>
			<td></td>
		</tr>
		<tr>
			<td>Manual Pipetting&#160; (10-100 &#181;L)</td>
			<td>Eppendorf</td>
			<td>3120000046</td>
			<td></td>
		</tr>
		<tr>
			<td>Manual Pipetting&#160; (20-200 &#181;L)</td>
			<td>Eppendorf</td>
			<td>3120000054</td>
			<td></td>
		</tr>
		<tr>
			<td>Manual Pipetting&#160; (100-1000 &#181;L)</td>
			<td>Eppendorf</td>
			<td>3120000062</td>
			<td></td>
		</tr>
		<tr>
			<td>Mounting Medium With DAPI&#160;- Aqueous, Fluoroshield</td>
			<td>abcam</td>
			<td>ab104139</td>
			<td></td>
		</tr>
		<tr>
			<td>Normal Human IgG Control</td>
			<td>R&#38;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&#160;X-100 solution</td>
			<td>Sigma Aldrich</td>
			<td>93443</td>
			<td></td>
		</tr>
		<tr>
			<td>TWEEN&#160;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 &#176;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...

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, ...

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.

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