Liquid Biopsy: CTC and ctDNA Analysis

Dual Isolation and Profiling of Circulating Tumor Cells and Circulating
Tumor DNA from a Single Liquid Biopsy Sample Using the Genesis
Cell Isolation System with Celselect Slides™ and Droplet Digital™ PCR
ProtocolAbstract
Recent advances and technology development in liquid biopsy have paved the way for noninvasive methods of cancer monitoringand diagnosis. Circulating tumor cells (CTCs) and circulating tumor DNA (ctDNA) are gaining more attention as promising biomarkers,
offering molecular insights into cancer progression, overall survival, and treatment efficacy.
In this context, we introduce a protocol that describes the dual isolation of ctDNA and CTCs from the same sample. This is facilitatedwith the Genesis Cell Isolation System and Droplet Digital PCR (ddPCR™), along with optimized blood component separation andcell-free DNA (cfDNA) enrichment steps. After sample preparation, the Genesis System with Celselect Slides enriches CTCs based on
size and deformability, and the enriched cells can be recovered for subsequent downstream analysis. Concurrently, Droplet Digital PCR
can be utilized to interrogate specific loci within cfDNA to determine the presence of a range of DNA aberrations present in ctDNA,
including single nucleotide variants and insertions and deletions.
This protocol using a model CTC-ctDNA sample serves as a starting point for specific cancer-associated mutation detection.
For example, EGFR mutations in ctDNA are a common biomarker in non–small-cell lung cancer (NSCLC) diagnostics. Their presenceor absence can shape NSCLC treatment strategies and influence patient prognoses. Additionally, analyzing CTCs from the same
sample can shed light on functional cancer studies and drug susceptibility assays.
A list of recommended materials, reagent preparations, and methods are provided, accompanied by example results from model
CTC-ctDNA samples. Users are recommended to select and validate their assays and cell systems with this protocol prior to use.
For a visual overview of the dual-isolation/profiling workflow steps, refer to Figure 1.

Part 1. Model sample preparation and plasma separation

Part 2. cfDNA extraction and ctDNA mutation
analysis with Droplet Digital PCR
Cell-free DNA (cfDNA)

Circulating tumor DNA (ctDNA)

Part 3. Rare cell enrichment/enumeration

A liquid sample containing
multiple circulating markers

Optimized preprocessing fordual isolation (CTCs and ctDNA)
Circulating tumor cells (CTCs)

Fig. 1. Overview of dual isolation of CTCs and cfDNA from a single liquid biopsy sample. The protocol is compatible with blood samples prepared in standard
BD Vacutainer EDTA Tubes (Becton, Dickinson and Company) or Cell-Free DNA BCT Tubes (Streck).

Bulletin 3599

Dual Isolation and Profiling of Circulating Tumor Cells and Circulating Tumor DNA from a Single Liquid Biopsy Sample
Using the Genesis Cell Isolation System with Celselect Slides and Droplet Digital PCR

Instruments and Materials

Sample Preparation of Model Blood Sample* and Plasma Separation
for Dual Isolation
In order to mimic blood samples from patients with lung cancer, amodel blood sample from a patient with lung cancer was preparedby spiking two model circulating markers, cancer cells (model ofCTCs) and fragmented genomic DNA containing EGFR mutations(model of ctDNA), into an untreated healthy donor’s whole blood
before any plasma separation or preprocessing steps.

Instruments■

 enesis Cell Isolation System (Bio-Rad Laboratories, Inc.,G
catalog #CEL20101)■

Q X200™ Droplet Generator (Bio-Rad, #1864002)■

Q X200 Droplet Reader (Bio-Rad, #1864003)

■

■

■

■

■

■

■

Q X200 AutoDG™ Droplet Digital PCR System(Bio-Rad, #1864100) (optional)■

PX1 PCR Plate Sealer (Bio-Rad, #1814000)
 1000 Touch Thermal Cycler with 96–Deep Well Reaction
C
Module (Bio-Rad, #1851197)■

2100 Bioanalyzer System (Agilent Technologies, Inc., #G2939BA)■■

 orvall ST 8 Small Benchtop Centrifuge
S(Thermo Fisher Scientific, #75007200)
 entrifuge 5810R 15 amp version (Eppendorf SE, #022625501)
C
with a rotor FA-45-6-30
Celselect Slides Enrichment Kit (Bio-Rad, #CEL80110)
 elselect Slides Enumeration Indirect Stain Kit
C(Bio-Rad, #CEL80111)
 dPCR Mutation Detection Assay: EGFR T790M
d(Bio-Rad, Assay ID #dHsaMDV2010019)■

Droplet Generation Oil for Probes (Bio-Rad, #1863005)■

Automated Droplet Generation Oil for Probes (Bio-Rad, #1864110)■

ddPCR Supermix for Probes (No dUTP) (Bio-Rad, #1863023)■

 tDNA model: Anchor Molecular multiplexed 23 ctDNA Standardcenzymatically fragmented (~145 bp band >95%)

Sample Preparation of Model Blood Sample from Patient with Lung Cancer(optional)*

 orvall Legend Micro 21R Microcentrifuge
S(Thermo Fisher Scientific Inc., #75002446)

Materials■

 TC model: A549 is a human non–small-cell lung cancer cell lineCwith the wild-type EGFR

1.

Prepare a cell stock suspension at a concentration of~10–20 cells/µl.

2.

Prepare a ctDNA standard solution at a concentration of
30 ng/μl.

3.

Spike 10 µl of cell stock solution into 4 ml of whole blood.

4.

Spike 2 µl of ctDNA stock solution into 4 ml of whole blood(this can lead to ~30 ng of ctDNA per 1 ml of plasma).

5.

Mix well with model CTC and ctDNA samples. Store thismodel blood sample from a patient with lung cancer on a
gentle blood rocker/shaker at room temperature until use.
If it is longer than 1 hr, store at 4°C.

* Required only for initial model sample testing. Alternatively, clinical samples withknown molecular pathology can be used.

Plasma Separation Using Two-Step Centrifugations (option A) G8 Cartridges for QX200 Droplet Generator
D(Bio-Rad, #1864008)

1.

Centrifuge either 4 ml of the model sample (see Sample
Preparation of Model Blood Sample from Patient with Lung
Cancer instructions) or 4 ml of actual clinical blood samples
at 2,000 rcf for 15 min.

■

Pierceable Foil PCR Plate Heat Seal (Bio-Rad, #1814040)■

ddPCR 96-Well Plates (Bio-Rad, #12001925)■

DNA LoBind Tubes, 1.5 ml (Eppendorf, #022431021)2.

 pplied Biosystems MagMAX Cell-Free DNA Isolation Kit
A(Thermo Fisher Scientific, #A29319) for cfDNA extraction (refer tothe MagMAX Cell-Free DNA Isolation Kit for additional materials
not supplied with the kit)

Upon first centrifugation, transfer the upper transparent yellow
plasma layer (~2 ml) to a sterile centrifuge tube.

3.

Shake the plasma sample mildly by hand for 15–20 seconds
and centrifuge the plasma sample at 16,000 rcf for 10 min.

 IAamp Circulating Nucleic Acid Kit (QIAGEN, #55114) forQ
cfDNA extraction (refer to the QIAamp Circulating Nucleic Acid
Handbook for additional materials not supplied with the kit)

4.

Upon second centrifugation, transfer the clear plasma layer(~2 ml) to a new sterile centrifuge tube. Store the clear plasma
layer in 4°C or in an ice bucket with ice until use.

 ultiplexed 23 ctDNA 3% AF (Anchor Molecular, #60100101)
M(optional)

5.

The leftover layer of blood cells (~2 ml) from step 2 will befurther diluted with 1x phosphate buffered saline (PBS)(for enrichment) or diluted fixing reagent (for enumeration).
See the Rare Cell Enrichment/Enumeration Workflow Using
the Genesis System with Celselect Slides section for details.
Store the leftover layer of blood cells on a gentle blood rocker/
shaker at room temperature until use. If it is longer than 1 hr,store at 4°C.

■

■

■

■


CytivaFicoll-Paque PLUS Media(Thermo Fisher Scientific, #17144002)

Note: Additional materials and equipment are needed to carryout the plasma separation and cfDNA extractions using the kitsmentioned here.

© 2023 Bio-Rad Laboratories, Inc.

2

Bulletin 3599

Dual Isolation and Profiling of Circulating Tumor Cells and Circulating Tumor DNA from a Single Liquid Biopsy Sample
Using the Genesis Cell Isolation System with Celselect Slides and Droplet Digital PCR

2.

Plasma Separation Using a Ficoll-Paque PLUS Media Solution (option B)

1.

To a 15 ml conical sterile centrifuge tube, add either 4 ml of
the model sample (see Sample Preparation of Model Blood
Sample from Patient with Lung Cancer instructions) or 4 mlof clinical sample blood. Then add 4 ml of 1x PBS to thesame tube.

2.

Mix the blood and PBS by inverting the tube several times.

3.

To a new 15 ml conical sterile centrifuge tube, add 4 mlof Ficoll-Paque PLUS Media solution.

4.

Carefully layer the diluted blood (from step 2) onto the
Ficoll-Paque PLUS Media solution. It is important, whenlayering the sample, to not mix the Ficoll-Paque PLUS Media
solution and the diluted blood sample.

5.

Centrifuge at 400 g for 30 min at 18°C while the brake is
set to 0.

6.

Upon centrifugation, transfer the upper layer (~6 ml)
containing diluted plasma and platelets to a new conical tube
using a sterile pipet, leaving the buffy coat layer containing
mononuclear cells undisturbed at the interface.

a. Label 15 ml conical tubes for each sample.
b. Pipet ~2 ml of processed, plasma-separated blood sample
into its respective 15 ml conical tube.
c. Add 6 ml of 1x PBS (blood sample from option A) or
4 ml of 1x PBS (blood sample from option B) and shakethe processed blood–PBS mixture vigorously by hand
15–20 seconds.
3.

In Running the Genesis System (see user manual), add 6–8 mlof sample to Inlet Funnel of each Celselect Slides Station.

4.

Follow the remaining steps in Running the Genesis System.

Celselect Slides Enumeration (direct/indirect)

1.

9.

b. Celselect Slides and Slide Station Set-Up
c. Priming the Celselect Slides
d. Preparing the Reagents and Loading Reagents Cartridge

Centrifuge the diluted plasma and platelets (~6 ml) at 16,000 rcf
for 10 min and transfer the clear plasma layer (~6 ml) to a newsterile centrifuge tube. Store the clear plasma layer at 4°C or inan ice bucket with ice until use.

2.

The layer of buffy coat (from step 7) will be further diluted
with 1x PBS (for enrichment) or diluted fixing reagent (forenumeration). See the Rare Cell Enrichment/Enumeration
Workflow Using the Genesis System with Celselect Slides
section for details. Store the leftover layer of blood cells on a
gentle blood rocker/shaker at room temperature until use. If it
is longer than 1 hr, store at 4°C.

b. For each sample, add 300 µl of fixing reagent to 2.7 ml of
1x PBS. Mix well.
c. Pipet ~2 ml of processed, plasma-separated blood sample
into its respective 15 ml conical tube.
d. Add 3 ml of diluted fixing reagent from step b to the~2 ml of preprocessed blood for each sample (final volume
is ~5 ml).
e. Incubate samples for 10 min on a rocker at room
temperature.
f. Remove samples from the rocker and add 3 ml of 1x PBS(for diluted fixing reagent + blood sample from option A)(final volume is ~8 ml) or 5 ml of 1x PBS (for diluted fixing
reagent + blood sample from option B) (final volume is~10 ml).

Celselect Slides Enrichment

Refer to the relevant sections of the Celselect Slides
Enrichment User Manual (bulletin 3337) to perform thefollowing steps of Celselect Slides enrichment.

g. Place blood sample on the test tube rocker until use.

a. Performing the Genesis System Self Test
b. Celselect Slides and Slide Station Set-Up

3.

In Running the Genesis System (see relevant user manual,
depending on the type of staining), add 8–10 ml of sample toInlet Funnel of each Celselect Slides Station.

4.

Follow the remaining steps in Running the Genesis System.

c. Priming the Celselect Slides
d. Preparing the Reagents Cartridge© 2023 Bio-Rad Laboratories, Inc.

In Preparing the Blood Sample (see relevant user manual,
depending on the type of staining), use either the blood
sample from step 5 in option A or step 9 in option B.
a. Label 15 ml conical tubes for each sample.

Rare Cell Enrichment/Enumeration Workflow Using theGenesis System with Celselect Slides
Either the sample from step 5 in option A or step 9 in option B
can be used for rare cell enrichment or enumeration.
1.

Refer to the Celselect Slides CTC Enumeration Direct
Staining User Manual (bulletin 3339) or Celselect Slides CTC
Enumeration Indirect Staining User Manual (bulletin 3338),
depending on the type of staining.
a. Performing the Genesis System Self Test

7. 	Carefully transfer the buffy coat layer containing mononucleatedcells (~2 ml) to a sterile centrifuge tube using a sterile pipet.
8.

In Preparing the Blood Sample (see user manual), use either the
blood sample from step 5 in option A or step 9 in option B.

3

Bulletin 3599

Dual Isolation and Profiling of Circulating Tumor Cells and Circulating Tumor DNA from a Single Liquid Biopsy Sample
Using the Genesis Cell Isolation System with Celselect Slides and Droplet Digital PCR

Cell-Free DNA Extraction and EGFR Mutation Detection Workflow
for Droplet Digital PCR
For cfDNA extraction, users can choose any circulating nucleic
acid kit or cfDNA extraction kit that is available on the market.
Here, two cfDNA extraction kits, MagMAX Cell-Free DNA Isolation
Kit and QIAamp Circulating Nucleic Acid Kit, were each tested byfollowing its standard protocol. Refer to each kit’s standard manual
for additional instruments or materials that are not supplied withthe kit.
■■■

Table 1. ddPCR reaction setup.

 sing the clear plasma sample from step 4 in option A or stepU
8 in option B, follow the instructions for manual cfDNA isolation
given in the MagMAX Cell-Free DNA Isolation Kit User Guide.
Either of the options for sample lysis (with or without PK) may
be used. The “Prepare cell-free plasma samples” step may beskipped, as the samples have already undergone centrifugation
at 16,000 rcf
 IAamp Circulating Nucleic Acid Kit: Refer to the QIAamp
Q
Circulating Nucleic Acid Handbook for extraction protocols.
Specifically, follow the Protocol: Purification of Circulating Nucleic
Acids from 1 ml, 2 ml, or 3 ml Serum or Plasma using the clear
plasma sample from step 4, option A. For the diluted plasma
sample from step 8, option B, follow the Protocol: Purification of
Circulating Nucleic Acids from 4 ml or 5 ml Serum or Plasma. If
needed, the diluted plasma sample can be aliquoted into morethan 2 tubes to follow the general protocols
 lution volume from each kit is flexible and can be adaptedEdepending on the requirements of downstream applications.
Elute and make the final cfDNA extraction volume less than
50 µl

1x

20x target (FAM) and wild-type(HEX) primers/probe1

1x

Restriction enzyme, diluted

1

2-5 U/reaction

Target sample and/or
DNase-free water

Variable

Variable

Total volume

20*

—

3.

Mix thoroughly by vortexing each tube. Centrifuge briefly toensure that all components are at the bottom of the reaction
tubes. Allow reaction tubes to equilibrate at room temperature
for about 3 minutes.

4.

Once the reaction mixtures are ready, load 20 μl of eachreaction mix into a sample well of a DG8 Cartridge followed
by 70 μl of Droplet Generation Oil for Probes into the oil wells,
according to the QX200 Droplet Generator Instruction Manual(D094813). For the Automated Droplet Generator, follow
instructions in the Automated Droplet Generator Instruction
Manual (D112916).

1.

After droplet generation with the QX200 Droplet Generator,
carefully transfer droplets into a clean ddPCR 96-Well Plateor remove the plate containing ddPCR droplets from theAutomated Droplet Generator. Seal the plate with the PX1 PCR
Plate Sealer.

2.

Proceed to thermal cycling (see protocol in Table 2) andsubsequent reading of droplets in the QX200 Droplet Reader.

Table 2. Recommended thermal cycling conditions for the C1000 Touch
Thermal Cycler with 96–Deep Well Reaction Module.

Thaw all components to room temperature. Mix thoroughly
by vortexing the tube to ensure homogeneity because aconcentration gradient may form during –20°C storage.
Centrifuge briefly to collect contents at the bottom of eachtube and store protected from light.

Cycling Step

Temperature,
°C

Time

Ramp Rate

Numberof Cycles

Enzyme activation95

10 min2°C/sec

1

Denaturation

94

30 sec2°C/sec

40

Annealing/extension

55

1 min

2°C/sec

40

Enzyme deactivation98

10 min2°C/sec

1

Hold (optional)

4

Infinite

1°C/sec

1

Data Acquisition and Analysis

Prepare samples at the desired concentration before setting
up the reaction mix according to the guidelines in Table 1. If
multiple samples are to be assayed using the same target andreference duplex assay, prepare a master reaction mix withoutsample template, dispense equal aliquots into the reaction
tubes, and add the sample template to each reaction tube as
the final step.

© 2023 Bio-Rad Laboratories, Inc.

Final Concentration

10

Thermal Cycling Conditions

Reaction Setup

2.

Volume per Reaction, µl

2x ddPCR Supermix for Probes(No dUTP)* For the Automated Droplet Generator, prepare 22 µl per well.

ctDNA EGFR Mutation Detection Workflow for Droplet Digital PCR
ddPCR Mutation Detection Assays are expertly designed
specifically for Droplet Digital PCR. The assays used in this protocol
consist of mutant-target and wild-type assays mixed and provided
in a single tube, with FAM targeting the mutant allele and HEX
targeting the wild-type allele. This assay is made up of a single
primer set plus two competitive probes.
1.

Component

4

1.

After thermal cycling, place the sealed 96-well plate in the
QX200 Droplet Reader.

2.

Open QX Manager Software Standard Edition to set up a newplate layout according to the experimental design. Refer to the
QX200 Droplet Reader and QX Manager Software Standard
Edition User Guide (10000107223).

3.

To add a plate in QX Manager Software, click the Add Platetab. The software should enable the Configure Plate button.
Click Configure Plate to open the Plate Configuration window.

Bulletin 3599

Dual Isolation and Profiling of Circulating Tumor Cells and Circulating Tumor DNA from a Single Liquid Biopsy Sample
Using the Genesis Cell Isolation System with Celselect Slides and Droplet Digital PCR

4.

The Configure Plate window opens the window features. Fill in
the plate and well information, and select the wells. Designate
the sample name, experiment type, supermix type (enter
ddPCR Supermix for Probes [No dUTP]), target name(s), andtarget (types). Select Reference Signal Ch1 for FAM and Signal
Ch2 for HEX.

CD45
(WBCs)

Cytokeratin(cancer cells )DAPI(nucleus staining)

20 µm

20 µm

20 µm

5.

Click Apply to load the wells and, when finished, click OK.

Fig. 3. A549 cancer cells and leukocytes captured in Celselect Slides after
following the indirect enumeration protocol using the Genesis System.
DAPI, 4',6-diamidino-2-phenylindole; WBCs, white blood cells.

6.

Once the plate layout is complete, click Run to begin thedroplet reading process. Select the appropriate dye set andrun options when prompted.

ctDNA EGFR T790M Mutation Detection Using Droplet Digital PCR

8.

After data acquisition, select samples in the well selector
under Analyze. Set the appropriate threshold for the ddPCR
Mutation Detection Assay. For more detailed information about
setting thresholds, refer to the Rare Mutation Detection Best
Practices Guidelines (bulletin 6628).

MAGMAX Cell-Free DNA Isolation Kit with Two-Step Centrifugation Technique

The concentration reported is copies/μl of the final
1x ddPCR reaction.

FAM amplitude

7.

2-D plots of ctDNA recovered from cell-free DNA extraction
are shown in Figure 4. Bio-Rad ddPCR EGFR T790M Mutation
Detection Assay was used. Two different cfDNA extraction
methods were compatible in this study and only the blood sample
with model ctDNA spiked shows positive droplets for EGFR T790M.

Results
Celselect Slides Enrichment/Enumeration (indirect)

7,000

7,000

6,000

6,000

5,000

5,000

4,000

4,000

3,000

3,000

2,000

2,000

1,000

1,000

0

Capture efficiency (Figure 2) and a representative image of A549
cancer cells captured in Celselect Slides and stained by CD45,
cytokeratin, and 4',6-diamidino-2-phenylindole (DAPI) (Figure 3) aredescribed here. In spite of additional plasma separation steps andliquid handlings, the Genesis System with Celselect Slides isolates
cancer cells spiked in whole blood with minimal cell loss (<6%).

0
0

500

1,000
1,500
2,000
HEX amplitude

2,500

Capture efficiency, %84%(standard)

79%

3,000

0

EGFR T790M 0 ng ctDNA per ml of plasma (control)

500

1,000
1,500
2,000
HEX amplitude

2,500

3,000

EGFR T790M 30 ng ctDNA per ml of plasma

FAM amplitude

QIAamp Circulating Nucleic Acid Kit Isolation with Two-Step Centrifugation Technique
7,000

7,000

6,000

6,000

5,000

5,000

4,000

4,000

3,000

3,000

2,000

2,000

1,000

100

EGFR T790M mutation

78%

1,000

0

0
0

500

1,000
1,500
2,000
HEX amplitude

2,500

EGFR T790M 0 ng ctDNA per ml of plasma (control)

80

EGFR T790M mutation

3,000

0

500

1,000
1,500
2,000
HEX amplitude

2,500

3,000

EGFR T790M 30 ng ctDNA per ml of plasma

Fig. 4. EGFR T790M DNA mutation in ctDNA enriched from whole blood. 2-D
amplitude plot of ctDNA spiked in blood in two different concentrations followed bya two-step centrifugation technique using MAGMAX Cell-Free DNA Isolation Kit and
QIAamp Circulating Nucleic Acid Kit. ctDNA (30 ng) was spiked into 4 ml of plasma
sample (right). High sensitivity of ddPCR Mutation Detection Assays provides precise
quantification of EGFR T790M mutation in the cell-derived ctDNA fragments obtained
from vendor. ctDNA, circulating tumor DNA.

60
40
20
0
None

Two-step cent. Ficoll-Paque(option A)(option B)
Plasma separation method

Fig. 2. Capture efficiency of A549 lung cancer cells spiked in whole blood
after plasma separation using the Genesis System with Celselect Slides. The
A549 cells were spiked in whole blood for a model CTC sample, which underwent
either two-step centrifugations or Ficoll-Paque PLUS Media solution–based plasma
separation. After plasma separation, the remaining portion of blood or buffy coatlayer containing mononuclear cells was run through the Genesis System with
Celselect Slides. The sample labeled “None” is a capture efficiency from standard
whole blood samples without any plasma separation. Cent., centrifugation.

© 2023 Bio-Rad Laboratories, Inc.

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Dual Isolation and Profiling of Circulating Tumor Cells and Circulating Tumor DNA from a Single Liquid Biopsy Sample
Using the Genesis Cell Isolation System with Celselect Slides and Droplet Digital PCR

For research use only. Not for use in diagnostic procedures.
Visit bio-rad.com/Celselect for more information.
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Bio-Rad Laboratories, Inc. in certain jurisdictions. All trademarks used herein are the property of their respective owner.
© 2023 Bio-Rad Laboratories, Inc.
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