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

Summary

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.

Abstract

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.

Introduction

Early and accurate detection of various diseases, including cancer, is the most important factor in establishing a treatment strategy^1,2,3,4. In particular, early detection of cancer is closely related to increased survival chances for the patient^5,6,7,8. 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 patients^2,9. 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 stages^6,10. 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 patients^11,12,13. 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 purity^13,14. Conventional centrifugation methods are commonly used, but they are difficult to handle and have low purity depending on the user'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 difficulties^16,17. 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'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.

Protocol

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

1. Collect 9 mL of whole blood into an EDTA or cfDNA-stable blood collection tube.
2. Mix well by flipping the tube up and down approximately 10 times.
3. Store the samples at room temperature (RT; for short-term storage) or 4 °C (for long-term storage). Do not freeze and thaw.

2. Device preparation

1. 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.
2. Select the sample mode to be used. Change the number of samples by pressing the arrow.

3. Device operation and sample collection

1. Loading of the LBx-1 cartridge
   1. Select the cartridge type on the touchscreen panel of the instrument. Change the number of samples by pressing the arrow button.
   2. 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.
   3. For a total of four cartridges, place the dummy cartridge in the empty space of the cartridge holder.
   4. Use the support wheel to mount the cartridge and tighten the lock nut to secure it.
   5. 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.
   6. Follow the message to open the door, remove the support wheel, and remove the valve-closed cartridge from the cartridge holder.
   7. 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.
   8. 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 µL of whole blood, which was separated before this step.
   9. 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.
   10. Look for the message on the screen accompanied by an alarm sound, which appears when the separation of plasma and buffy coat is complete.
   11. Stop the alarm by opening the door or pressing the STOP button. Open the door, remove the cartridge, and place it on the table.
   12. 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.
2. Loading the LBx-2 cartridge
   1. Select the cartridge name on the touchscreen panel of the instrument. Change the number of samples by pressing the arrow button.
   2. Open the door and insert the cartridges to be used in order of the number on the cartridge holder.
   3. For a total of four cartridges, place the dummy cartridge in the empty space of the cartridge holder.
   4. 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.
   5. 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.
   6. 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.
   7. 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).
   8. 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.
   9. 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).
   10. 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.
   11. 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.
   12. Stop the alarm by opening the door or pressing the STOP button. Open the door, remove the cartridge, and place it on the table.
   13. 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.
3. Loading the FAST-auto cartridge
   1. Select the cartridge on the touch screen panel of the instrument. Change the number of samples by pressing the arrow button.
   2. 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.
   3. Use the support wheel to mount the cartridge and tighten the lock nut to secure it.
   4. 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.
   5. Follow the message to open the door, remove the support wheel, and remove the valve-closed cartridge from the cartridge holder.
   6. 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.
   7. 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.
   8. 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.
   9. 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.
   10. 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.
   11. 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.
   12. 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.

4. Maintenance of the system

1. Preparation for cleaning and disinfection of the instrument
   1. Clean all the accessible surfaces of the instrument and the accessories once a week using ethanol and dried tissue, and also immediately when contaminated.
   2. Clean the bowl and rotor shaft regularly using alcohol (ethanol and isopropanol) or alcohol-based disinfectants.
2. Cleaning and disinfecting the instrument
   NOTE: For general troubleshooting and other notes on the machine, refer to Supplementary Table 2.
   1. Switch off the instrument using the main power switch. Disconnect the power plug from the power supply.
   2. 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.
   3. Check the rotor shaft for damage. Inspect the instrument for corrosion and damage.
   4. Connect the instrument to the power supply only if it is fully dry inside and out.
3. 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.

Representative Results

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.

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. Please click here to view a larger version of this figure.

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. Please click here to view a larger version of this figure.

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 µm. Please click here to view a larger version of this figure.

Supplementary Table 1: Composition of the final volume for density gradient solution use. Please click here to download this File.

Supplementary Table 2: General issues and cautions for maintenance. Please click here to download this File.

Discussion

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 patient^2,4,5,10,20. 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 cancer^{21,22,23,24,25}. 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 µm²⁷. In another study, the mean size of the measured WBC was 9 µm. On the other hand, the minimum size of isolated CTCs is 16 µm, and the average size is 30 µm²⁸. 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.

Materials

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