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SYSMEX INOSTICS
en
ZR150148
OncoBEAM™ RAS CRC Kit
OBMRASIVD.R2
IN VITRO TEST | For In Vitro Diagnostic Use.
Glossary of Symbols
Manufacturer
Keep away from sunlight
In Vitro Diagnostic Medical Device
Lot Number
Catalog Number
Danger: Acute Toxicity
Warning: Other Health Effects
Danger: Flammable Substances
Danger: Corrosives
Do not re-use
Caution
Consult instructions for use
Contains sufficient for <n> tests
Used by
Temperature limit
Recent data from numerous prospective and retrospective
studies have demonstrated that a more comprehensive analysis
of KRAS and NRAS codons beyond KRAS 12 & 13 can also identify
mutations in patients that are unlikely to benefit from anti-EGFR
therapy.7-12 In the OPUS and CRYSTAL studies an additional 15 to
26% of patients, whose tumors were previously classified as KRAS
codons 12 & 13 wild-type, were found to harbor less common
mutations in either NRAS or KRAS.7,8 Patients with these
infrequent RAS mutations demonstrated similar, and in some
cases, worse outcomes to anti-EGFR therapy as patients with
KRAS codon 12 and 13 mutations. According to meta-analysis
across all studies involving a comprehensive analysis of RAS
mutations beyond KRAS codons 12 & 13, an additional 10 to 15%
of patients were identified who would not benefit from anti-EGFR
therapy.3 Based on the data in these studies, ESMO, NCCN, and
ASCO guidelines were updated in 2014/2015 to reflect the need
for broader RAS testing across the full spectrum of KRAS and
NRAS mutations to identify a significant number of patients for
which anti-EGFR therapy is not effective.13,14,21 The OncoBEAM
RAS CRC Kit offers a targeted RAS mutation analysis aligned with
current clinical practice guidelines.
Proprietary Name
OncoBEAM RAS Colorectal Cancer (CRC) Kit
Intended Use
The OncoBEAM RAS qualitative digital PCR based test is intended
for the detection of gene mutations in KRAS codons 12, 13,
59, 61, 117, 146 and NRAS codons 12, 13, 59, 61, 117, 146
extracted from Stage IV colorectal cancer patient plasma. The kit
is intended to aid the clinician in determining potential benefit
of anti-epidermal growth factor receptor (EGFR) therapy to
colorectal cancer patients.
Summary and Explanation
KRAS testing for mutations in codons 12 & 13 of Exon 2 was
established as a clinical practice guideline in 2009 for metastatic
colorectal cancer (mCRC) patients eligible for treatment with
anti-EGFR therapy.1,2 KRAS codon 12 & 13 mutations occur in
approximately 40% of patients with metastatic colorectal cancer.
These KRAS mutations are associated with a lack of response
to treatment with anti-EGFR monoclonal antibodies.3-6 Initial
clinically approved assays were designed to evaluate 7 mutations
in codons 12 and 13 of the KRAS oncogene.
OBMRASIVD.R2
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The OncoBEAM RAS CRC Kit is for the qualitative detection of
RAS (KRAS and NRAS) mutations in plasma of Stage IV colorectal
cancer patients using BEAMing digital PCR technology that
employs beads-based amplification in water-in-oil emulsions and
allele-specific hybridization followed by flow cytometry.
The OncoBEAM RAS CRC Kit detects mutations in codons 12, 13,
59, 61, 117 and 146 of the KRAS and NRAS oncogenes against a
background of wild-type genomic DNA.
4.
Mutation detection using allele-specific hybridization probes
that are reverse complementary to the known wild-type
and mutant sequences. Differentiation of beads, carrying
copies of the mutant or the wild-type alleles is done via flow
cytometry analysis.
Figure 1. Workflow Process
Table 1. List of KRAS/NRAS Mutations detected by
OncoBEAM RAS CRC Kit 15
Gene/Amplicon
Nucleotide Change
Codon Position
G>S
g34c
G>R
g34t
KRAS 2
g35a
12
g35c
G>V
g38a
13
G>D
g175a
59
A>T
a182t
Q>L
a182g
Q>R
a183c
61
a183t
KRAS 4A
KRAS 4B
a351c
a351t
g436a
c437t
Q>H
117
146
A>T
A>V
G>S
12
G>C
G>D
g35c
G>A
g35t
G>V
g38a
G>R
13
g38t
g175a
A>T
Q>K
a182g
a182t
G>D
G>V
59
c181a
Q>R
61
Q>L
a183c
Q>H
a183t
Q>H
g351c
NRAS 4
K>N
G>R
g37c
NRAS 3
K>N
g34c
g34t
g351t
g436a
The workflow process shown in Figure 1 is described as follows:
1. Blood Collection/Plasma Preparation:
Plasma specimens to be tested with the OncoBEAM RAS
CRC Kit are collected in the Streck Cell-Free DNA BCT device
according to the Streck's instructions for use (IFU). Refer to
the Plasma Preparation section of this IFU.
2. DNA Extraction:
DNA extraction from plasma samples is done by using
Qiagen's QIAAmp® Circulating Nucleic Acid Kit. Refer to
DNA Extraction section in this IFU.
3. PCR Amplification:
a. Multiplex PCR Pre-Amplification:
The isolated plasma DNA eluates are subjected to
Multiplex PCR in 6 replicates using the Multiplex Primer
Mix, the Multiplex Master Mix and Multiplex PCR DNA
Polymerase which are provided with the OncoBEAM
RAS CRC Kit for use on a ThermoFisher's Veriti Dx
thermal cycler instrument. Multiplex PCR is performed
to co-amplify the regions of interest under high-fidelity
PCR conditions.
Primers are targeting a region within KRAS Exon 2, a
region within KRAS Exon 3, two regions within KRAS
Exon 4, one region within NRAS Exon 2, one region
within NRAS Exon 3, and a region within NRAS Exon 4.
Non‑allele specific primers bind within these regions and
are thereby ensuring unbiased amplification of wild-type
as well as mutant molecules.
b. Pooling and Dilution:
After multiplex PCR, the 6 replicate PCR reactions from
each eluate are manually pooled in a new PCR plate and
diluted with specific fixed dilution factors defined in the
IFU. Dilution is done manually using 1x TE Buffer
(10 mM Tris-HCl, 0.1 mM EDTA, pH 8.0).
4. Emulsion PCR Amplification:
Diluted Multiplex PCR product is used as input to the
Emulsion PCR, which requires the EmulsiFIRE, Emulsion
Beads, the Emulsion DNA Polymerase and the specific
Emulsion PCR Mastermixes which are provided with the
OncoBEAM RAS CRC Kit, for use on ThermoFisher's Veriti Dx
thermal cycler instrument.
Preparation of the emulsion requires use of an electronic
pipette properly programmed resulting in formation of
water in oil emulsion with an optimized number of water
compartments. Emulsion PCRs are exon specific with exon
specific primer pairs. These emulsion PCR primers bind
outside the targeted sites, thereby ensuring amplification of
Q>H
g34a
g35a
NRAS 2
G>C
G>D
G>A
g35t
KRAS 3
Amino Acid Change
g34a
117
146
K>N
K>N
A>T
The OncoBEAM RAS CRC Kit is to be used by trained personnel in
a professional molecular diagnostic laboratory environment.
Principles of the Procedure
The OncoBEAM RAS CRC Kit constitutes a ready-to-use in vitro
nucleic acid amplification assay for the qualitative detection of a
total of 34 mutations and differentiation of the 12 codons of the
human KRAS and NRAS genes using the BEAMing technology. The
kit is used with a system of instruments, software and reagents
and consists of multiple workflow processes.
Main process steps are:
1. Specimen preparation to extract cell-free ctDNA from
human plasma.
2. Preamplification in a multiplex approach to enrich
target DNA.
3. Emulsion-based digital PCR in combination with specific
amplification for each gene exon on magnetic beads.
OBMRASIVD.R2
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Analysis of Flow Data and Report:
Analysis of the flow cytometry data fcs-file is performed using
FCS Express V5.0. For analysis, raw data are loaded into a
template with fixed analysis settings.
Generated PDF summary sheets contain analysis template
specific information about measurement validity (sample or
control "Valid" or "Invalid") and for samples also information
about mutation status ("Mutation Detected" or "No
Mutation Detected"). The absolute number of mutant beads
has to exceed a minimum level, in order to ensure that the
measurement is within an acceptable range. Measurement
validity criteria include internal control for evaluation of
adequate sample DNA concentration. External control
measurements confirm the validity of test results. Results are
reported for each valid sample (ie. for a sample with 12 valid
measurements) cumulatively as “Mutation Detected” in case
one or more of the 12 measurements resulted in a positive
result or as “No Mutation Detected” for samples for which all
12 measurements were found to be negative.
NOTE:OncoBEAM RAS CRC specific thermal cycler temperature
programs will be provided to the customer and installed
on their equipment prior to first use. Programs will be
provided in order to perform the following validated
workflow process sections:
• Multiplex PCR
• Emulsion PCR
• Hybridization
Additionally, OncoBEAM RAS CRC specific flow cytometry
acquisition settings will be provided to the customer as
well and installed on the Sysmex Partec Cyflow Cube6i
Flow Cytometer prior to first use, in order to perform the
Data Acquisition process section.
each of the 7 exons of the multiplex PCR from wild-type and
mutant molecules. Amplicons, which harbor 2 codons are
prepared in 2 parallel reactions.
A water-in-oil emulsion is created with each aqueous
microdroplet containing an individual fragment of DNA. This
allows millions of compartmentalized PCRs to be performed
in parallel in one single test tube resulting in beads coated
with thousands of copies of DNA that are identical to the
original template DNA molecule, either mutant or wild-type.
5. Breaking and Hybridization:
After emulsion PCR, emulsions are broken releasing magnetic
beads coated with amplified PCR products which are then
recovered using a magnet plate. Bead-bound PCR products
are denatured, leaving only 1 DNA strand bound to the
beads. Beads with single stranded DNA are purified with a
magnet plate. Breaking and DNA denaturation is achieved
using breaking buffers 1 and 2, which are provided with the
OncoBEAM RAS CRC Kit, and requires use of an electronic
pipette properly programmed according to specifications in
this IFU. The allele-specific hybridization step is performed
using codon-specific probe mixes and hybridization buffer,
which are provided with the OncoBEAM RAS CRC Kit,
and electronic pipette properly programmed according to
specifications in this IFU. Probe hybridization is achieved
after completing hybridization program using ThermoFisher's
Veriti Dx thermal cycler instrument.
Each of the 12 emulsion PCR products is hybridized with a
mixture of 3 different fluorescent labeled oligonucleotide
probe types (1 distinct probe mixture for each codon to
be detected). To distinguish mutant from wild-type coated
beads, allele-specific fluorescent probes complementary
to the known wild-type and mutant sequences are
simultaneously added to the beads for hybridization. One of
the oligonucleotide probes, the "universal probe" binds to
the target amplicon outside of the target region and is used
to distinguish between beads that contain PCR products and
beads without PCR products. The 2 other oligonucleotide
probes are wild-type and mutant specific probes that target
the respective allele and label the beads according to their
mutational status.
Each of the 3 probe types within each probe mix is labeled
with a specific fluorescent reporter molecule.
For each of the 12 codons, all probes detecting target
mutations are provided in the same mixture, together with
the respective wild-type probe and the universal probe.
After hybridization, magnetic beads are washed and resuspended with PBS using an electronic pipette properly
programmed according to specifications in this IFU, resulting
in an acceptable concentration for flow cytometry readout.
6. Flow Cytometry:
The beads are detected using a flow cytometry instrument,
a detection platform that measures fluorescent signals.
Forward and side scatter are used to differentiate between
single beads and clusters of multiple beads and gating is used
to select the single bead population. Single beads are then
analyzed for the fluorescent dye indicating the presence of
PCR products (universal signal) and gating is used to select
single beads containing PCR amplified products. Single beads
containing PCR amplified products are then subjected to the
analysis of the two fluorescent dyes indicating the presence
of beads with wild-type, mutant, or both (mixed) PCR
products. Fixed instrument settings are defined in a template.
The fluorescent intensities of the 3 dyes are recorded for each
bead and stored in a .FCS file for each measurement.
OBMRASIVD.R2
7.
Reagents
Storage and Handling
The Sysmex Inostics OncoBEAM RAS CRC
Kit catalog number ZR150101 must be
stored at – 15 to – 30°C when not in use.
The Sysmex Inostics OncoBEAM RAS CRC
Kit catalog number ZR150102 must be
stored at 2 to 8°C when not in use.
The Sysmex Inostics OncoBEAM RAS CRC
Kit catalog number ZR150103 must be
stored at 15 to 25°C when not in use.
Shipping Conditions
The Sysmex Inostics OncoBEAM CRC RAS Kit item ZR150101
ships on dry ice.
The Sysmex Inostics OncoBEAM CRC RAS Kit items ZR150102
and ZR150103 ship at 15 to 25°C.
General Handling Precautions
Ensure that the laboratory is kept at room temperature while
performing assay workflow.
Do not eat, drink, or smoke in laboratory areas.
Perform equipment maintenance according to
manufacturer's instructions.
Decontaminate and dispose of all reagents, specimens, and
associated supplies in accordance with government regulations
for your location.
3
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For accurate and reproducible results, it is essential to
avoid contamination with foreign DNA, especially PCR
products from previously run plates. The most common
source of DNA contamination is the amplified products from
previous experiments.
4.
Materials Provided
Store at – 15 to – 30°C.
NOTE:Reagents may not be mixed between different
OncoBEAM RAS CRC Kit lots.
NOTE:For reagents stored at – 15 to – 30°C, two freeze/thaw
cycles are allowed. The external controls are single use!
1.
Composition: • Tris EDTA Buffer
• Carrier RNA
• double stranded synthetic DNA wildtype KR4B
• double stranded synthetic DNA
mutant KR4B
Positive Control Amplicon KRAS Exon 2
Catalog
ZR150112
Number:
Quantity:
0.15 mL per vial (5 vials included)
Storage:
5.
Store at – 15 to – 30°C.
Composition: • Tris EDTA Buffer
• Carrier RNA
• double stranded synthetic DNA wildtype KR2
• double stranded synthetic DNA
mutant KR2
2.
Composition: • Tris EDTA Buffer
• Carrier RNA
• double stranded synthetic DNA wildtype NR2
• double stranded synthetic DNA
mutant NR2
Positive Control Amplicon KRAS Exon 3
Catalog
ZR150113
Number:
Quantity:
0.15 mL per vial (5 vials included)
Storage:
6.
Composition: • Tris EDTA Buffer
• Carrier RNA
• double stranded synthetic DNA wildtype KR3
• double stranded synthetic DNA
mutant KR3
Positive Control Amplicon NRAS Exon 3
Catalog
ZR150117
Number:
Quantity:
0.15 mL per vial (5 vials included)
Storage:
Store at – 15 to – 30°C.
Composition: • Tris EDTA Buffer
• Carrier RNA
• double stranded synthetic DNA wildtype NR3
• double stranded synthetic DNA
mutant NR3
Positive Control Amplicon KRAS Exon 4A
Catalog
ZR150114
Number:
Quantity:
0.15 mL per vial (5 vials included)
Storage:
7.
Store at – 15 to – 30°C.
Composition: • Tris EDTA Buffer
• Carrier RNA
• double stranded synthetic DNA wildtype KR4A
• double stranded synthetic DNA
mutant KR4A
OBMRASIVD.R2
Positive Control Amplicon NRAS Exon 2
Catalog
ZR150116
Number:
Quantity:
0.15 mL per vial (5 vials included)
Storage:
Store at – 15 to – 30°C.
Store at – 15 to – 30°C.
3.
Positive Control Amplicon KRAS Exon 4B
Catalog
ZR150115
Number:
Quantity:
0.15 mL per vial (5 vials included)
Storage:
Positive Control Amplicon NRAS Exon 4
Catalog
ZR150118
Number:
Quantity:
0.15 mL per vial (5 vials included)
Storage:
Store at – 15 to – 30°C.
Composition: • Tris EDTA Buffer
• Carrier RNA
• double stranded synthetic DNA wildtype NR4
• double stranded synthetic DNA
mutant NR4
4
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8.
No Template Control
Catalog
ZR150119
Number:
Quantity:
0.15 mL per vial (5 vials included)
Storage:
11. Multiplex PCR DNA Polymerase
Catalog
Number:
Quantity:
Storage:
ZR150122
0.55 mL per vial (1 vial included)
Store at – 15 to – 30°C.
Store at – 15 to – 30°C.
Composition: • Tris EDTA Buffer
• Carrier RNA
9.
Composition: • Tris-HCI (pH 7.4 at 25°C)
• EDTA
• DTT
• KCI
• stabilizers
• BSA
• Triton X-200
• 50% Glycerol
NOTE: Before first use, prepare 5 aliquots of 105 µL in
separate 1.5 mL tubes. Use only 1 aliquot per
run (plate).
Multiplex PCR Master Mix
Catalog
ZR150120
Number:
Quantity:
3.50 mL per vial (5 vials included)
Storage:
Store at – 15 to – 30°C.
Composition: •
•
•
•
RNAse-free and DNAse-free distilled water
PCR Buffer
Magnesium Chloride
Deoxynucleotide Triphosphates
12. Emulsion PCR Master Mix KRAS Exon 2
10. Multiplex PCR Primer Mix
Catalog
Number:
Quantity:
Storage:
Multiplex PCR DNA Polymerase is Thermo Scientific™ Phusion™ Hot Start HighFidelity DNA Polymerase.
This product is manufactured by Thermo Fisher Scientific, Inc.
Catalog
Number:
Quantity:
Storage:
ZR150121
1.45 mL per vial (5 vials included)
ZR150123
0.28 mL per vial (5 vials included)
Store at – 15 to – 30°C.
Store at – 15 to – 30°C.
Composition: •
•
•
•
•
•
•
•
Composition: •
•
•
•
•
RNAse-free and DNAse-free distilled water
Primer KR2
Primer KR3
Primer KR4A
Primer KR4B
Primer NR2
Primer NR3
Primer NR4
RNAse-free and DNAse-free distilled water
PCR Buffer
Magnesium Chloride
Deoxynucleotide Triphosphates
Primer KR2
13. Emulsion PCR Master Mix KRAS Exon 3
Catalog
Number:
Quantity:
Storage:
ZR150124
0.28 mL per vial (5 vials included)
Store at – 15 to – 30°C.
Composition: •
•
•
•
•
OBMRASIVD.R2
5
RNAse-free and DNAse-free distilled water
PCR Buffer
Magnesium Chloride
Deoxynucleotide Triphosphates
Primer KR3
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18. Emulsion PCR Master Mix NRAS Exon 4
14. Emulsion PCR Master Mix KRAS Exon 4A
Catalog
Number:
Quantity:
Storage:
Catalog
Number:
Quantity:
Storage:
ZR150125
0.14 mL per vial (5 vials included)
ZR150129
0.28 mL per vial (5 vials included)
Store at – 15 to – 30°C.
Store at – 15 to – 30°C.
Composition: •
•
•
•
•
RNAse-free and DNAse-free distilled water
PCR Buffer
Magnesium Chloride
Deoxynucleotide Triphosphates
Primer KR4A
Composition: •
•
•
•
•
15. Emulsion PCR Master Mix KRAS Exon 4B
Catalog
Number:
Quantity:
Storage:
19. Emulsion PCR DNA Polymerase
ZR150126
Catalog
Number:
Quantity:
Storage:
0.14 mL per vial (5 vials included)
Store at – 15 to – 30°C.
Composition: •
•
•
•
•
ZR150130
0.13 mL per vial (5 vials included)
Store at – 15 to – 30°C.
RNAse-free and DNAse-free distilled water
PCR Buffer
Magnesium Chloride
Deoxynucleotide Triphosphates
Primer KR4B
Composition: •
•
•
•
•
•
16. Emulsion PCR Master Mix NRAS Exon 2
Catalog
Number:
Quantity:
Storage:
RNAse-free and DNAse-free distilled water
PCR Buffer
Magnesium Chloride
Deoxynucleotide Triphosphates
Primer NR4
ZR150127
Tris-HCl
KCI
EDTA
55% Glycerol
Tween-20
Nonidet P40
20. Emulsion PCR Beads
Catalog
Number:
Quantity:
Storage:
0.28 mL per vial (5 vials included)
Store at – 15 to – 30°C.
ZR150131
0.55 mL per vial (1 vial included)
Store at 2 to 8°C.
Composition: •
•
•
•
•
RNAse-free and DNAse-free distilled water
PCR Buffer
Magnesium Chloride
Deoxynucleotide Triphosphates
Primer NR2
Composition: • RNAse-free and DNAse-free distilled water
• Magnetic Beads coupled with primer
• ProClin
21. EmulsiFIRE
17. Emulsion PCR Master Mix NRAS Exon 3
Catalog
Number:
Quantity:
Storage:
Catalog
Number:
Quantity:
Storage:
ZR150128
0.28 mL per vial (5 vials included)
ZR150144
10 mL per bottle (5 bottles included)
Store at 15 to 25°C.
Store at – 15 to – 30°C.
Composition: •
•
•
•
•
OBMRASIVD.R2
Composition: • Oil
• Emulgent
• Tegosoft DEC
RNAse-free and DNAse-free distilled water
PCR Buffer
Magnesium Chloride
Deoxynucleotide Triphosphates
Primer NR3
6
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22. Breaking Buffer 1 without Alcohols
Catalog
Number:
Quantity:
Storage:
26. Hybridization Probe Mix 2
ZR150145
Catalog
Number:
Quantity:
Storage:
50 mL per bottle (1 bottle included)
ZR150133
0.06 mL per vial (1 vial included)
Store at 2 to 8°C, in the dark.
Store at 15 to 25°C.
Composition: • Tris EDTA Buffer
• ProClin
• Fluorescent-labelled Probes KR2 Codon 13
Composition: • Water per analysis (p.A.)
• SDS solution
• Sodium Chloride
• Tris-HCl Buffer
• Triton X-100
• EDTA
NOTE: Before first use, add alcohols according to the
instructions in this package insert.
27. Hybridization Probe Mix 3
Catalog
Number:
Quantity:
Storage:
ZR150134
0.06 mL per vial (1 vial included)
23. Breaking Buffer 2 without Alcohols
Catalog
Number:
Quantity:
Storage:
Store at 2 to 8°C, in the dark.
ZR150146
Composition: •
•
•
•
80 mL per bottle (1 bottle included)
Store at 15 to 25°C.
28. Hybridization Probe Mix 4
Composition: • Water per analysis (p.A.)
• Sodium Hydroxide
• SDS solution
• Sodium Chloride
• Triton X-100
• EDTA
NOTE: Before first use, add alcohols according to the
instructions in this package insert.
Catalog
Number:
Quantity:
Storage:
ZR150147
29. Hybridization Probe Mix 5
Catalog
Number:
Quantity:
Storage:
22 mL per bottle (2 bottles included)
Composition: •
•
•
•
ZR150136
0.06 mL per vial (1 vial included)
Store at 2 to 8°C, in the dark.
Tris-HCl Buffer
EDTA
Water per analysis (p.A.)
Tetramethylammonium chloride
Composition: • Tris EDTA Buffer
• ProClin
• Fluorescent-labelled Probes KR4A Codon
117
25. Hybridization Probe Mix 1
ZR150132
30. Hybridization Probe Mix 6
Catalog
Number:
Quantity:
Storage:
0.06 mL per vial (1 vial included)
Store at 2 to 8°C, in the dark.
ZR150137
0.06 mL per vial (1 vial included)
Store at 2 to 8°C, in the dark.
Composition: • Tris EDTA Buffer
• ProClin
• Fluorescent-labelled Probes KR2 Codon 12
OBMRASIVD.R2
0.06 mL per vial (1 vial included)
Composition: • Tris EDTA Buffer
• ProClin
• Fluorescent-labelled Probes KR3 Codon 61
Store at 15 to 25°C.
Catalog
Number:
Quantity:
Storage:
ZR150135
Store at 2 to 8°C, in the dark.
24. Hybridization Buffer
Catalog
Number:
Quantity:
Storage:
Tris EDTA Buffer
ProClin
Blocker
Fluorescent-labelled Probes KR3 Codon 59
Composition: • Tris EDTA Buffer
• ProClin
• Fluorescent-labelled Probes KR4B
Codon 146
7
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31. Hybridization Probe Mix 7
Catalog
Number:
Quantity:
Storage:
36. Hybridization Probe Mix 12
ZR150138
Catalog
Number:
Quantity:
Storage:
0.06 mL per vial (1 vial included)
ZR150143
0.06 mL per vial (1 vial included)
Store at 2 to 8°C, in the dark.
Store at 2 to 8°C, in the dark.
Composition: • Tris EDTA Buffer
• ProClin
• Fluorescent-labelled Probes NR2 Codon 12
Composition: • Tris EDTA Buffer
• ProClin
• Fluorescent-labelled Probes NR4
Codon 146
32. Hybridization Probe Mix 8
Catalog
Number:
Quantity:
Storage:
Non-Reagent Laboratory Equipment
ZR150139
37. Tube strips
Catalog Number: ZR150149
Quantity: 10 strips and caps included
0.06 mL per vial (1 vial included)
General Materials
Store at 2 to 8°C, in the dark.
Materials Required But Not Provided
Laboratory Equipment
• Streck Cell-Free DNA Blood Collection Tubes, 10.0 mL volume,
Streck #218996 for 6 tube pack, Streck #218997 for 100
tube box
NOTE: Streck Cell-Free DNA blood collection tubes are provided
together with the specimen shipping box. These tubes
may be used for samples that are being sent out for
testing or for in-house laboratory testing.
• BD Vacutainer® K2 EDTA Blood Collection Tubes with
Lavender HemogardTM Closure, 16 × 100 mm × 10.0 mL
volume, Becton Dickinson, #367525
NOTE: BD Vacutainer K2 EDTA blood collection tubes may only
be used for in-house laboratory testing of samples due to
stricter restrictions for plasma preparation timeframe.
• Aerosol-resistant sterile pipette tips with filters 20 µL
• Aerosol-resistant sterile pipette tips with filters 200 µL
(Mettler Toledo, #17014963)
• Aerosol-resistant sterile pipette tips with filters 1000 µL
• Serological Pipette, 10 mL, sterile
• Serological Pipette, 5 mL, sterile
• Serological Pipette, 25 mL, sterile
• 10 mL centrifuge tubes
• 15 mL centrifuge tubes
• Tube strips plus cap
• 4 mL cryogenic vial
• Freezer storage boxes
• Tubes 3.5 mL
• Tubes 1.5 mL
• Tubes 5 mL
• Deepwell Plate (500 µL, DNA LoBind)
• Super Plate Skirted PCR, Thermo Fisher AB-2400 (required)
• Disposable reagent reservoirs (25 mL)
• PCR plate sealing film (Aluminium)
• PCR plate sealing foil (transparent)
• 96 well rack
• Tube chain rack
• Tube rack
• Ice bucket
• Film Applicator
• Mini-Cooler
• Protective Glasses
• Protective Sleeves
Composition: • Tris EDTA Buffer
• ProClin
• Fluorescent-labelled Probes NR2 Codon 13
33. Hybridization Probe Mix 9
Catalog
Number:
Quantity:
Storage:
ZR150140
0.06 mL per vial (1 vial included)
Store at 2 to 8°C, in the dark.
Composition: • Tris EDTA Buffer
• ProClin
• Fluorescent-labelled Probes NR3 Codon 59
34. Hybridization Probe Mix 10
Catalog
Number:
Quantity:
Storage:
ZR150141
0.06 mL per vial (1 vial included)
Store at 2 to 8°C, in the dark.
Composition: • Tris EDTA Buffer
• ProClin
• Fluorescent-labelled Probes NR3 Codon 61
35. Hybridization Probe Mix 11
Catalog
Number:
Quantity:
Storage:
ZR150142
0.06 mL per vial (1 vial included)
Store at 2 to 8°C, in the dark.
Composition: • Tris EDTA Buffer
• ProClin
• Fluorescent-labelled Probes NR4 Codon 117
OBMRASIVD.R2
8
DocuSign Envelope ID: 825DA068-1DE5-471B-B6D1-989C098CA476
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Protective Disposable Shoes
Protective Gloves
Protective Coats
Centrifuge for 1.5/2 mL tubes, capable of 20817 × g,
fixed‑angle rotor
Centrifuge for 15/50 mL tubes, capable of 7197 × g,
fixed angle rotor
Centrifuge for Plasma Preparation, capable of 6000 × g for
Streck Cell Free DNA BCT or capable of either 3000 × g or
6000 × g for BD Vacutainer K2 EDTA tubes, swing bucket rotor,
smooth acceleration and breaking profiles, with adaptors for
10 mL blood and conical tubes
Vortex
Minicentrifuge capable of ≤ 2000 × g
Pipettor 1000 µL
Pipettor 200 µL
Pipettor 20 µL
Pipettor 10 µL
Multi 12-channel pipettor 300 µL
Multi 12-channel pipettor 200 µL
Multi 12-channel pipettor 20 µL
Pipettor 300 µL
Pipettor 5 - 50 mL
Waterbath + Holder for 50 mL tubes
Thermometer
Heatblock
Freezer, – 15 to – 30°C
Refrigerator, 2 to 8°C
Deep freezer, – 70°C or colder
DNA workstation/PCR cabinet
Rainin E12-200XLS+ (electronic pipettor), Mettler Toledo,
#17013800
QIAvac Connecting System, Qiagen, #19419
QIAvac 24 Plus System, Qiagen, #19413
QIAamp Circulating NA Kit, Qiagen, #55114
Vacuum Pump (230 V, 50 Hz), Qiagen, #84020
Veriti Dx 96-well Thermal Cycler, 0.2 mL tubes block format,
ThermoFisher Scientific, #4452300
Alpaqua 96S Super Magnet Plate, Alpaqua, #A001322
Sysmex Partec CyFlow® Cube6i Flow Cytometer, #CY-S-3065R
Stopwatch
Fume Hood (strongly recommended)
Class II Biological Safety Cabinets (strongly recommended)
Ethanol ≥ 99.8%, p.a.
1-Propanol ≥ 99.8%, p.a.
2-Propanol ≥ 99.8%, p.a.
Mineral Oil
2-Butanol ≥ 99%
1x Phosphate Buffer Saline solution (PBS)
1x TE buffer, low EDTA (pH 8.0)
Sheath Fluid, Sysmex Partec, #04-4007
Decontamination Solution, 250 mL Sysmex Partec, #04-4010
Citrate Cleaning Concentrate Solution, #04-4015
Ultra-violet light
8 Peak Beads Concentrate, Sysmex Partec, #05-4025
Red Fluorescent Particles Concentrate,
Sysmex Partec, #05‑4024
Calibration Beads; High Concentration (Blue), 30 mL
Sysmex Partec, #05-4023
Sheath filter, Sysmex Partec, #04-004-1000
OBMRASIVD.R2
• Container for Cleaning and Decontamination solution, Sysmex
Partec, #04-2015-02
• Rack for 96 well plates for CyFlow® Robby 6/8
Autoloading Station, #04-2016
Warnings and Precautions
In Vitro Diagnostic Medical Device
The Hybridization Buffer contains Tetramethylammonium
chloride (TMAC). The following warning statements are
associated with the Hybridization Buffer reagent:
Danger
Hazard-determining components of labeling:
Tetramethylammonium chloride
H301 Toxic if swallowed.
P270
Do not eat, drink or smoke when using
this product.
P301 + P310IF SWALLOWED: Immediately call a
POISON CENTER or doctor.
P330
Rinse mouth.
P405
Store locked up.
P501
Dispose of contents/container in accordance
with local and national regulations.
The EmulsiFIRE mixture contains bis(2-ethylhexyl) carbonate.
The following warning statements are associated with the
EmulsiFIRE reagent:
Warning
Hazard-determining components of labeling:
bis(2-ethylhexyl) carbonate
H315
Causes skin irritation.
P280
Wear protective gloves/eye protection.
P332+P313 If skin irritation occurs: Get medical
advice/attention.
CAUTION: Breaking Buffers 1 & 2 mixture contains
sodium-dodecyl-sulfate. Breaking Buffers 1 & 2 will also be
mixed with alcohols 2-Butanol and 1-Propanol in the
laboratory by customer. The following warning statements
are associated with the Breaking Buffer reagents:
Danger
Hazard-determining components of labeling:
2-Butanol and 1-Propanol
H226
Flammable liquid and vapor.
H318
Causes serious eye damage.
H335
May cause respiratory irritation.
H336
May cause drowsiness or dizziness.
P210
Keep away from heat, hot surfaces, sparks,
open flames and other ignition sources.
No smoking.
P261
Avoid breathing vapors/spray.
P280
Wear protective gloves/protective clothing/
eye protection/face protection.
P305+P351 IF IN EYES: Rinse cautiously with water for
+P338
several minutes. Remove contact lenses, if
present and easy to do. Continue rinsing.
P310
Immediately call a POISON CENTER/doctor.
P501
Dispose of contents/container in accordance
with local and national regulations.
NOTE: Safety Data Sheets (SDS) for all reagents provided in the
kits are available upon request.
9
DocuSign Envelope ID: 825DA068-1DE5-471B-B6D1-989C098CA476
Specific Measures
Precautions for Safe Handling
Avoid contact with skin and eyes. Avoid inhalation of vapor
or mist.
Conditions for Safe Storage, Including Any Incompatibilities
Keep container tightly closed in a dry and well-ventilated place.
Containers which are opened must be carefully resealed and kept
upright to prevent leakage.
• Clean PCR workspaces with alcohol-based disinfectant (such
as Bacillol® Plus or equivalent) followed by cleaning with
product designed for removing nucleic acids and nucleases
(such as PCR Clean™ or equivalent) before and after use.
• Decontaminate PCR workspaces and labware (pipets, tube
racks, etc.) with ultraviolet (UV) light before use. Ensure UV
bulbs are cleaned regularly from accumulating residue to
ensure UV radiation is effective.
• Use only aerosol-resistant sterile pipette tips with filters (lotcertified, RNase-, DNase- and pyrogen-free).
• Use only PCR-grade reagents and tubes.
• Do not open (peel back) aluminum foil seal from PCR plates
containing amplified products. Use pipette tips to pierce the
aluminum foil within a particular PCR plate row or column and
discard the pipette tips. Then, use new pipette tips to aspirate
and transfer the reactions.
• Keep only 1 specimen tube or reagent tube open at a time.
• Keep tubes containing reagents or samples open a minimal
amount of time.
• To prevent contamination of multiple use reagent solutions,
prepare working aliquots according to instructions and
minimize direct pipetting.
Safety and Contamination Precautions
Reagent Handling Precautions
First Aid Measures
General advice: Consult a physician. Show this safety data sheet
to the doctor in attendance. Move out of dangerous area.
If inhaled: If breathed in, move person into fresh air. If not
breathing, give artificial respiration. Consult a physician.
In case of skin contact: Wash off with soap and plenty of water.
Take victim immediately to hospital. Consult a physician.
In case of eye contact: Rinse thoroughly with plenty of water for at
least 15 minutes and consult a physician.
If swallowed: Never give anything by mouth to an unconscious
person. Rinse mouth with water. Consult a physician.
Handling and Storage
Follow the precautions listed below to maintain a laboratory work
environment free of DNA contamination and to ensure laboratory
personnel safety:
• Wear appropriate personal protective equipment (PPE)
throughout the procedure when handling reagents and
specimens. Wear lab coat (disposable is preferred) and
disposable powder-free gloves at all times when working
in the pre-PCR and post-PCR laboratories. Change gloves
frequently between handling specimens and reagents
and after contact of the outside of the gloves with skin to
prevent contamination.
• Wear protective eyeglasses during hybridization and
wash steps for protection against Tetramethylammonium
chloride (TMAC).
• Wear disposable shoe covers and disposable arm protection
sleeves (required in the pre-PCR lab and recommended in the
post-PCR lab).
• Remove and discard personal protective equipment when
exiting pre- and post-PCR laboratory areas.
• Handle all specimens as potentially infectious material. If
a spill occurs, it is recommended to clean the affected area
first with detergent/disinfectant and water and then with
0.5% sodium hypochlorite (bleach) solution prepared using
deionized water.
NOTE: Commercial liquid house household bleach (e.g.
Clorox brand) typically contains sodium hypochlorite
at a concentration of 5.25%. A 1:10 dilution of
household bleach will produce a 0.5% sodium
hypochlorite solution.
• Separate the workspaces used for pre-PCR and post-PCR
laboratory operations and adhere to unidirectional workflow
from ‘clean’ (pre-amplification) to ‘dirty’ (post-amplification
areas) on any given day.
• Have dedicated equipment (including dedicated pipettors),
supplies, reagents, biohazard waste containers and lab
manuals in each work area and never exchange these
materials between pre- and post-PCR work areas. Color coding
or labeling of equipment, supplies and reagents to identify
those that belong to a particular area is recommended.
OBMRASIVD.R2
Follow the precautions listed below to ensure proper use and
disposal of reagents and to avoid contamination of reagents:
• Check upon arrival of kit that all reagents delivered on dry ice
are still frozen (except enzymes).
• Check expiration dates and storage conditions printed on the
OncoBEAM RAS CRC Kit labels of all reagent kit components
and store according to label temperature specifications.
• Do not use expired or incorrectly stored reagents.
• Prepare reagents according to provided instructions.
• Reagents are intended to be used only with the other reagents
in the same kit.
• Reagents from different lots may not be pooled
or interchanged.
• Record open date and mark tubes after each use to
ensure reagents are not used past open expiration date or
recommended number of freeze-thaw cycles for reagents
stored frozen.
• Avoid contamination of reagents by changing gloves
frequently. Change gloves always between handling reagents
and specimens.
• Dispose of unused reagents and waste according to country,
federal, state and local environmental regulations.
• Use caution when handling Hybridization Buffer which
contains Tetramethylammonium chloride (TMAC). TMAC is
a hazardous chemical in case of eye contact, skin contact,
ingestion, or inhalation.
–– Use adequate general or local exhaust ventilation to
keep TMAC airborne concentrations low. No respiratory
protection required where adequate ventilation conditions
exist. If airborne concentration is high, a respirator
is recommended.
–– Pipette tips that come in contact with TMAC must be
discarded in TMAC solid waste bag, must be segregated
from other solid waste, and disposed according to country,
federal, state and local environmental regulations.
–– Discard Hybridization Buffer in TMAC liquid waste
container, store container tightly closed, and
dispose according to country, federal, state and local
environmental regulations.
10
DocuSign Envelope ID: 825DA068-1DE5-471B-B6D1-989C098CA476
NOTE:If BD Vacutainer K2 EDTA and Streck Cell-Free DNA
BCT specimens are combined into one centrifuge,
centrifuge tubes at 15 to 25°C for 10 minutes at 6000
(±150) × g using a swing-out or fixed angle rotor.
7. Transfer plasma (supernatant) to a fresh 10 mL centrifuge
tube without disturbing the cell pellet by pipetting along the
tube wall.
CAUTION: Leave a residual volume of about 300 µL (~7 mm) on
the bottom of the tube to avoid contaminating the
plasma with cells.
8. Gently mix plasma by pipetting up and down 5 times using a
serological pipette.
9. Transfer at least 3 mL of the plasma into a 4 mL cryogenic vial.
10. If not directly proceeding with Purification of Circulating
DNA from Plasma steps, immediately place plasma cryogenic
vials in an upright position into freezer at ≤ – 70°C for storage
(up to a maximum of 24 months).
11. Record time of freezing and storage temperature.
Specimen Collection and Processing
Specimen Collection and Processing
Whole blood may be collected in Streck Cell-Free DNA BCT or
BD Vacutainer K2 EDTA blood collection tubes with lavender
Hemogard closure. Streck Cell-Free DNA BCT may be used
for samples that are being sent out for testing or for in-house
laboratory testing. BD Vacutainer K2 EDTA blood collection tubes
may only be used for in-house laboratory testing of samples due
to stricter restrictions for plasma preparation timeframe.
1. Collect 2 × 10 mL of blood per patient by venipuncture into
Streck Cell-Free DNA BCT or BD Vacutainer K2 EDTA blood
collection tubes with lavender Hemogard closure.
NOTE:Consult instructions for use for the Streck Cell-Free
DNA BCT (www.streck.com) or the BD Vacutainer K2
EDTA blood collection tubes (www.bd.com) for steps 2
through 4.
2. Fill each tube completely (until blood flow stops).
NOTE: Overfilling or underfilling of tubes will result in an
incorrect blood to additive ratio.
3. Immediately mix each tube by gentle inversion 8 to 10 times.
NOTE: One inversion is a complete turn of the wrist (180°)
and back.
4. Ensure that tubes are labeled and kept at ambient temperature.
NOTE: Do not refrigerate or freeze blood samples.
5. Record date and time of blood draw.
Purification of Circulating DNA from Plasma
Use the Qiagen QIAamp Circulating Nucleic Acid Kit reagents
to perform the purification of circulating DNA. Use the DNA
purification from plasma work order template to record
relevant information.
Preparation
12. If samples have been stored frozen, let them thaw and bring
to 15 to 25°C.
13. Set the water bath temperature to 60°C and verify the
temperature of the water with an external thermometer.
14. Set the heat block temperature to 56°C and verify the
temperature of the blocks with an external thermometer.
15. Set up the QIAvac 24 Plus as described in QIAamp Circulating
Nucleic Acid Kit Handbook.
16. Turn on the vacuum pump (set to –800 to –900 mbar) before
starting the work.
17. Equilibrate Buffer AVE to 15 to 25°C for elution.
18. Ensure that Buffer ACB, Buffer ACW1, and Buffer ACW2 have
been prepared according to the description below.
19. Prepare Carrier RNA in AVE buffer according to the
description below.
Preparation of Reagents and Buffers
Buffer ACB
NOTE: Preparation only required when using a new kit.
20. Before use, add 200 mL isopropanol (100%) to 300 mL buffer
ACB concentrate to obtain 500 mL Buffer ACB. Mix well after
adding isopropanol.
Buffer ACW1
NOTE: Preparation only required when using a new kit.
21. Before use, add 25 mL ethanol (96 to 100%) to 19 mL buffer
ACW1 concentrate to obtain 44 mL Buffer ACW1. Mix well
after adding ethanol.
Buffer ACW2
NOTE: Preparation only required when using a new kit.
22. Before use, add 30 mL ethanol (96 to 100%) to 13 mL buffer
ACW2 concentrate to obtain 43 mL Buffer ACW2. Mix well
after adding ethanol.
Carrier RNA
NOTE: Preparation only required when using a new kit. If carrier
RNA has already been dissolved, take a frozen aliquot out
of the freezer and bring to 15 to 25°C.
Procedure
Streck Cell-Free DNA BCT: Start plasma processing no later than 3
days after the time of the blood draw.
BD Vacutainer K2 EDTA tubes: Start plasma processing no later
than 4 hours after the time of the blood draw.
Plasma processing steps must be carried out as quickly as possible
without pause between individual steps. Handle all samples as
potentially infectious material. Use the Plasma Preparation work
order template to record all relevant information.
Plasma Preparation
1.
2.
3.
4.
5.
6.
Check blood collection tube type and corresponding date and
time of blood collection to ensure blood samples are valid:
≤ 3 days from collection to plasma preparation for Streck
Cell-Free DNA BCT or ≤ 4 hours from collection to plasma
preparation for BD Vacutainer K2 EDTA tubes.
Centrifuge blood collection tubes at 15 to 25°C for 10
minutes at 1600 (± 150) g using a swing bucket rotor. If
possible, switch off the centrifuge brake.
Record plasma processing date and time at start of
first centrifugation.
Visually check plasma fraction (supernatant) and
record observations.
NOTE: Highly hemolytic samples (reddish plasma) must
be rejected.
Transfer plasma (supernatant) from both tubes to one 10 mL
centrifuge tube without disturbing the cellular layer. Residual
plasma amount over leukocyte layer should be at least 500 µL.
For plasma samples from Streck Cell-Free DNA BCT,
centrifuge the plasma in 10 mL centrifuge tube at 15 to 25°C
for 10 minutes at 6000 (± 150) × g using a swing-out or fixed
angle rotor. For plasma samples from BD Vacutainer K2 EDTA
tubes, centrifuge tube at 15 to 25°C for 10 minutes at either
3000 (± 150) × g or 6000 (± 150) × g using a swing-out or fixed
angle rotor.
OBMRASIVD.R2
11
DocuSign Envelope ID: 825DA068-1DE5-471B-B6D1-989C098CA476
23. Add 1550 μL buffer AVE into the tube with 310 μg of
lyophilized carrier RNA. Mix by pipetting up and down.
24. Split into 75 μL aliquots in 1.5 mL tubes, label them and store
at –15 to –30°C freezer.
NOTE:Do not freeze-thaw aliquots more than 3 times.
25. Add carrier RNA dissolved in Buffer AVE (as described in
steps 23 and 24) to Buffer ACL according to Table 2 and mix
by gently inverting the tube 10 times.
38.
39.
Table 2. Volumes of Buffer ACL and Carrier RNA
(Dissolved in Buffer AVE) Required for
Processing 3 mL Plasma Samples
No of samples
ACL Buffer [mL]
40.
Carrier RNA in AVE Buffer [μL]
1
2.6
5.6
2
5.3
11.3
3
7.9
16.9
4
10.6
22.5
5
13.2
28.1
6
15.8
33.8
41.
42.
43.
Plasma Centrifugation Before DNA Extraction
26. For plasma samples from Streck Cell-Free DNA BCT, spin
down cryogenic vials containing plasma (brought to 15 to
25°C before) at 1000 × g using a fixed angle rotor, and stop
centrifugation once speed is reached. For plasma samples
from BD Vacutainer K2 EDTA tubes, spin down cryogenic vials
containing plasma for 5 minutes at 7198 × g using a fixed
angle rotor.
DNA Extraction
27. Pipet 300 μL Proteinase K into a 50 mL centrifuge tube for
each plasma sample.
28. Add 3 mL of plasma to the 50 mL tube containing
Proteinase K.
NOTE: Samples with < 3 mL of plasma must be rejected. Only
open the tubes from one test subject at a time. Take
care not to pipette the pellet and leave about 30 μL of
plasma in the tube.
29. Add 2.4 mL of Buffer ACL (containing carrier RNA) to each
plasma sample.
30. Close the tube cap and mix by pulse vortexing (vortexing in
short intervals) for 30 seconds. Then proceed immediately to
the next step to start the lysis incubation.
31. For plasma samples from Streck Cell-Free DNA BCT, incubate
at 60°C (± 1°C) in a water bath for 60 (± 2) minutes. For
plasma samples from BD Vacutainer K2 EDTA tubes, incubate
at 60°C (± 1°C) in a water bath for 30 (± 2) minutes.
32. Take the tube out of the waterbath, unscrew the cap and add
5.4 mL of Buffer ACB to the lysate in each tube.
33. Close the tube cap again and mix thoroughly by pulse
vortexing for 15 to 30 seconds.
34. Incubate the lysate-Buffer ACB mixture for 5 (± 1) minutes
on ice.
35. In the meantime place the labeled QIAamp Mini columns into
the VacConnector on the QIAvac 24 Plus and insert 20 mL
tube extenders (extension adapters) into the open QIAamp
Mini columns.
36. Turn on the vacuum pump (set to –800 to –900 mbar) while
the main vacuum valve remains closed.
37. After incubation on ice carefully apply the lysate–Buffer ACB
mixture from step 34 into the tube extender of the QIAamp
Mini column.
OBMRASIVD.R2
44.
45.
46.
47.
48.
49.
50.
51.
52.
53.
54.
55.
56.
12
NOTE: To avoid contamination do not perform work over the
column extender of other samples when transferring
the lysate.
Open the main vacuum valve and let the lysates pass through
the columns completely.
Close main vacuum valve (while vacuum stays on) and release
the vacuum pressure from the QIAvac 24 Plus to 0 mbar.
Carefully remove and discard the tube extenders.
NOTE:To avoid contamination, be careful when removing the
tube extenders so as to not perform work above other
sample tubes.
Apply 600 μL of Buffer ACW1 to the QIAamp Mini columns
using a fresh pipette tip for each sample.
Leave the lid of the column open and open the vacuum valve
to let Buffer ACW1 flow through the columns completely.
Close the main vacuum valve (while vacuum stays on) and
release the vacuum pressure from the QIAvac 24 Plus to
0 mbar.
Apply 750 μL of Buffer ACW2 to the QIAamp Mini columns
using a fresh pipette tip for each sample.
Leave the lid of the columns open and open the vacuum valve
to let Buffer ACW2 flow through the columns completely.
Close the main vacuum valve (while vacuum stays on) and
release the vacuum pressure from the QIAvac 24 Plus to
0 mbar.
Apply 750 μL of ethanol (96 to 100%) to the QIAamp Mini
columns using a fresh pipette tip for each sample.
Leave the lid of the columns open and open the vacuum valve
to let ethanol flow through the columns completely.
Close the main vacuum valve and switch off the vacuum pump.
Close the lid of the QIAamp Mini columns. Remove them
from the vacuum manifold, and discard the VacConnector.
NOTE:Avoid touching the rim of the columns or lids. Change
gloves if the rim of the columns or lids is touched
accidentally to avoid cross-contamination.
Place the QIAamp Mini columns into clean 2 mL collection
tubes, and centrifuge at 20,000 × g for 3 (± 0.5) minutes.
Place the QIAamp Mini columns into new 2 mL collection
tubes. Discard the used 2 mL tubes.
Open the lids and incubate the assembly within heat
block at 56°C (± 1°C) for 10 (± 1) minutes to dry the
membrane completely.
NOTE:Residual ethanol may interfere with downstream
BEAMing reactions. Ensure recommended drying time
is met.
Place the QIAamp Mini columns in clean 1.5 mL elution tubes
and discard the 2 mL collection tubes. Ensure that elution
tubes are labeled with sample ID.
NOTE:Only use elution tubes provided with the Qiagen
QIAamp Circulating Nucleic Acid Kit. Using tubes
other than those recommended may influence
downstream BEAMing reactions.
Carefully apply 70 μL of Buffer AVE to the center of the
QIAamp Mini membrane (without touching the membrane)
using a fresh pipette tip for each sample.
Close the lids and incubate at room temperature for
3 (± 0.5) minutes.
Centrifuge at 20,000 × g for 1 minute to elute the nucleic acids.
DocuSign Envelope ID: 825DA068-1DE5-471B-B6D1-989C098CA476
57. Carefully apply again 70 μL of Buffer AVE to the center of the
66. Using a multichannel pipette, aliquot 65 µL of template
QIAamp Mini membrane. Close the lids and incubate at room
temperature for 3 (± 0.5) minutes. Centrifuge at 20,000 × g for
1 minute to elute the nucleic acids.
NOTE:Do not vary from using 2 × 70 µL AVE buffer for
elution. Only recommended volume will ensure
sufficient eluate volume of 123 μL for downstream
BEAMing procedure.
58. Discard the QIAamp Mini columns.
59. Close 1.5 mL elution tubes tightly and store at 2 to 8°C (when
continuing within 24 hours) or at –15 to –30°C (up to a
maximum of 8 days).
Multiplex PCR
Use Multiplex PCR work order template to record
relevant information:
60. Take the following reagents and solutions out of the freezer
(pre-PCR Lab) and let them thaw at room temperature
(15 to 25°C).
• Multiplex PCR Master Mix
• Multiplex PCR Primer Mix
• Positive Controls (PC1 to PC7)
• No Template Control (NTC)
NOTE: Multiplex PCR DNA Polymerase (MEnz) does not
require thawing. Keep it in the freezer until ready
to use. Before first use, prepare 5 aliquots of 105 µL
in separate 1.5 mL tubes. Use only 1 aliquot per
run (plate).
61. Vortex Multiplex PCR Master Mix, Multiplex PCR Primer Mix,
and controls (PCs and NTC) for 5 seconds after thawing and
spin down with centrifuge for 2 seconds
62. Do not vortex Multiplex PCR DNA Polymerase (MEnz), as this
may inactivate the enzyme. Gently mix, then spin down with
centrifuge for 2 seconds.
NOTE:Multiplex PCR DNA Polymerase is viscous. To ensure
adequate pipetting of required enzyme volume
aspirate slowly.
63. Set up the necessary amount of Multiplex PCR Master Mix
(MMx), Multiplex PCR Primer Mix (MPx), and Multiplex PCR
DNA Polymerase (MEnz) for all samples in 5 mL tube(s),
according to Table 3. Mix reagents by pipetting up and down
10 times.
NOTE: If more than 6 samples are required, a 2nd plate will be
needed and will be run independently of the first plate.
67.
68.
69.
70.
71.
72.
Emulsion PCR
Use Emulsion work order template to record relevant information:
73. Remove Emulsion PCR Master Mix vials from freezer and let
thaw at room temperature.
NOTE: Emulsion PCR DNA Polymerase does not require
thawing. Keep at – 15 to – 30°C until ready to use.
74. Fill a conical tube with the required volume of 1x TE buffer,
low EDTA (pH 8.0) according to Table 4. Ensure 1x TE buffer is
equilibrated to room temperature before use.
Table 4. 1x TE Buffer, Low EDTA (pH 8.0)
Required Volumes
Number of Samples Tested
TE Buffer
Table 3. Multiplex PCR Master Mix Preparation
1871 µL 2079 µL 2286.9 µL 2495 µL 2703 µL 2911 µL
Multiplex PCR
Primer Mix (MPx)
772.2 µL
858 µL
943.8 µL
59.4 µL
Final Volume (sum)
2703 µL 3003 µL 3303 µL 3604 µL 3904 µL 4204 µL
Reaction tube size
5 mL
66 µL
5 mL
72.6 µL
5 mL
79.2 µL
5 mL
85.8 µL
5 mL
2
3
4
5
6
5 mL
6.5 mL
8 mL
9 mL
10.5 mL
12 mL
entered into the electronic pipettor, according to Table 5, and
check that battery power is at least 50%:
Table 5. Electronic Pipettor Specifications
(Abbreviations defined)a
1030 µL 1115 µL 1201 µL
Multiplex PCR DNA
Polymerase (MEnz)
[2U/µL]
1
75. Ensure the following electronic pipette program has been
Amount of samples
(1 sample =
1 sample 2 samples 3 samples 4 samples 5 samples 6 samples
6 replicates),
+1NTC +1NTC +1NTC +1NTC +1NTC +1NTC
with excess
+7PC
+7PC
+7PC
+7PC
+7PC
+7PC
Multiplex PCR
Master Mix (MMx)
+ Multiplex PCR master mix per well (total of 6 replicates
per sample) into a 96-well semi-skirted SuperPlate PCR plate
(ThermoFisher AB-2400). Use Multiplex PCR work order
template to ensure right position of samples and controls.
NOTE: Position the multichannel pipette vertically and
transfer first 65 μL of sample/master mix and NTC/
master mix from the first tube strip into the PCR plate
(6 wells per sample and 6 wells for NTC) as shown
in the Multiplex PCR work order template. Then,
transfer 65 μL of positive control/master mix from
the second tube strip into the PCR plate (6 wells per
positive control) as shown in the Multiplex PCR work
order template.
Transfer 3.5 mL of mineral oil into a disposable reservoir.
Cover each well with 20 µL of mineral oil. Seal plate with
adhesive aluminium foil. Ensure by visual inspection that all
wells are entirely covered by the foil and foil is not damaged.
Carry the plate from the pre-PCR lab to the cycler.
Do not exceed 15 minutes.
Place 96-well plate in PCR machine, close the cycler lid, and
run the Multiplex PCR program.
NOTE:Multiplex PCR program is set up by
Applications Specialist.
After the run, print PCR report and carefully remove
Multiplex PCR plate from the cycler.
Store Multiplex PCR plate at 2 to 8°C in the post-PCR
laboratory until further processing (up to a maximum of 48
hours). For longer term storage refer to step 78.
Program Name
92.4 µL
5 mL
64. For each sample and control aliquot 287 µL of Multiplex PCR
Master Mix into tube strips.
65. Mix with 123 µL of template (eluates of samples, no template
control or positive controls) per tube and mix by pipetting up
and down 10 times.
Specification
E
Aspirate volume (µL)
70
Aspirate speed
6
Dispense volume (uL)
70
Dispense speed
6
Mixing volume (µL)
80
Mixing cycles
30
Mixing speed
6
a:
E = Emulsion
NOTE:Program is set up by Applications Specialist.
OBMRASIVD.R2
13
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Pooling
76. Carefully pierce the foil with the 200 µL multichannel pipette
tips without touching the sample/oil layer. Use new pipette
tips for every new row or column.
77. Move new pipette tips through the oil layer down to the
bottom of the well, then move pipette up 1 mm to aspirate
the reaction mix. Take 15 µL from each of the 6 reactions per
sample and per control and pool reactions into the wells of a
new PCR plate according to pool plate layout in Emulsion PCR
work order template. Mix the wells for each of the pooled
reactions by pipetting up and down 10 times with a 200 µL
multi-channel pipette.
78. Seal the Multiplex PCR plate with aluminium adhesive foil
and store it in the refrigerator at 2 to 8°C for short term up to
a maximum of 48 hours. For longer term storage of the sealed
Multiplex PCR plate, store at – 15 to – 30°C up to a maximum
of 4 months.
Final Dilution
79. Transfer 1x TE buffer, low EDTA (pH 8.0) equilibrated at room
temperature to disposable reagent reservoir.
80. Transfer the appropriate volume of 1x TE buffer, low EDTA
(pH 8.0) into a 96-deep-well PCR plate (500 µL capacity, DNA
LoBind), according to Figure 2.
NOTE:For first dilution of PCs add 190 µL of TE buffer into
wells A12 to G12. Do not add TE buffer into well H12.
81. Using a multichannel pipette, add 10 µL of the sample and
NTC template from pool plate into column 1 (wells A1 to G1).
Then add 10 µL of positive controls into column 12 (wells
A12 to G12) of the deep-well dilution plate. Carefully mix by
pipetting up and down 10 times. Transfer 50 µL of PC7 first
dilution in well G12 to well H12. Refer to Figure 2.
82. Perform specific final dilutions using volumes listed in
Figure 2, for samples and NTC first (wells A3 to G10) and
then for positive controls (wells H3 to H10) by mixing 20 µL
of first dilution (from columns 1 and 12) to each final dilution
column (columns 3 to 10). Carefully mix by pipetting up and
down 10 times.
Preparation of Emulsion Working Mix (EWMX)
(Emulsion PCR Master Mix/Beads/Emulsion PCR DNA Polymerase)
84. Vortex Emulsion PCR Master Mix vials (7 vials total; 1 vial for
each amplicon) for 5 seconds after thawing. Spin down with
centrifuge for 2 seconds to ensure that no liquid remains at
the lid.
85. Vortex the beads thoroughly and make sure that all beads
have been re-suspended by flicking against the bottom of the
tube. Spin down with centrifuge for 2 seconds to ensure that
no liquid remains at the lid.
86. Gently mix the Emulsion PCR DNA Polymerase (EmEnz). Spin
down with centrifuge for 2 seconds to ensure that no liquid
remains at the lid. Do not vortex the Emulsion PCR DNA
Polymerase (EmEnz), as this may inactivate the enzyme.
NOTE:Emulsion PCR DNA Polymerase is viscous. To ensure
adequate pipetting of required enzyme volume
aspirate slowly.
87. Prepare each Emulsion Working Mix (EWMX) according
to the corresponding Table 6. Add reagents in the same
order indicated in the tables. Mix by pipetting up and down
10 times. To minimize subsequent pipetting steps, transfer
Emulsion Working Mixes into labeled tube strip.
NOTE: Mix gently to avoid foaming of the Emulsion
Working Mixes.
Table 6. Preparation of Emulsion Working Mixes
(Abbreviations defined)a
1 Sample + Controls
EWMX_1 EWMX_2 EWMX_3 EWMX_4 EWMX_5 EWMX_6 EWMX_7
EWMX 1 rxn (KR2)
(KR3) (KR4A) (KR4B) (NR2) (NR3) (NR4)
no of rxn
80 µL 80 µL 330 µL 280 µL 80 µL 130 µL 30 µL 80 µL
Pool DNA 10 µL
1st
dil
DNA
2
4
5
6
7
8
9
10 11 12
S1
S1
S1
S1
S1
S1
S1
PC1
B
S2
S2
S2
S2
S2
S2
S2
S2
S2
PC2
C
S3
S3
S3
S3
S3
S3
S3
S3
S3
PC3
D
S4
S4
S4
S4
S4
S4
S4
S4
S4
PC4
E
S5
S5
S5
S5
S5
S5
S5
S5
S5
PC5
F
S6
S6
S6
S6
S6
S6
S6
S6
S6
PC6
G
NTC
NTC NTC NTC NTC NTC NTC NTC NTC
PC7b
PC1
PC7
PC7b
1st dil
KR_2 KR_3 KR_4A KR_4B NR_2 NR_3 NR_4 NR_4
1st dil
1:20
12/13 59/61 117 146 12/13 59/61 117 146
1:100 1:100 1:350 1:300 1:100 1:150 1:50 1:100
1:20
PC5
PC6
PC7
8
8
105.6
105.6
105.6
Beads
(µL)
0.8
6.4
6.4
3.2
3.2
6.4
6.4
6.4
EmEnz
(µL)
1.0
8.0
8.0
4.0
4.0
8.0
8.0
8.0
Sum
(µL):
15.0
120.0
120.0
60.0
60.0
120.0
120.0
120.0
120
120
60
60
120
120
120
no of rxn
3
PC4
8
52.8
10 µL
S1
PC3
4
52.8
EWMX_1 EWMX_2 EWMX_3 EWMX_4 EWMX_5 EWMX_6 EWMX_7
EWMX 1 rxn (KR2)
(KR3) (KR4A) (KR4B) (NR2) (NR3) (NR4)
S1
PC2
4
105.6
190 µL
A
H
8
105.6
2 Samples + Controls
20 µL 20 µL 20 µL 20 µL 20 µL 20 µL 20 µL 20 µL
1
8
13.2
Total
volume
Figure 2.Volume Amounts Required for TE Buffer and
DNA Template (Abbreviations defined)a
TE 190 µL
1
EM 1-7
(µL)
1
10
10
5
5
10
10
10
EM 1-7
(µL)
13.2
132.0
132.0
66.0
66.0
132.0
132.0
132.0
Beads
(µL)
0.8
8.0
8.0
4.0
4.0
8.0
8.0
8.0
EmEnz
(µL)
1.0
10.0
10.0
5.0
5.0
10.0
10.0
10.0
Sum
(µL):
15.0
150.0
150.0
75.0
75.0
150.0
150.0
150.0
150
150
75
75
150
150
150
Total
volume
3 Samples + Controls
EWMX_1 EWMX_2 EWMX_3 EWMX_4 EWMX_5 EWMX_6 EWMX_7
EWMX 1 rxn (KR2)
(KR3) (KR4A) (KR4B) (NR2) (NR3) (NR4)
1
12
12
6
6
12
12
12
EM 1-7
(µL)
13.2
158.4
158.4
79.2
79.2
158.4
158.4
158.4
a: S = Sample, PC = Positive Control, NTC = No Template Control, KR = KRAS Exon,
NR = NRAS Exon
b: Transfer 50 μL of PC7 first dilution in well G12 to well H12 after adding DNA
template and mixing
Beads
(µL)
0.8
9.6
9.6
4.8
4.8
9.6
9.6
9.6
EmEnz
(µL)
1.0
12.0
12.0
6.0
6.0
12.0
12.0
12.0
83. Seal the pool plate with aluminum like adhesive foil and store
Sum
(µL):
15.0
180.0
180.0
90.0
90.0
180.0
180.0
180.0
180
180
90
90
180
180
180
Codon
Dilution
no of rxn
it in the refrigerator at 2 to 8°C up to a maximum of 7 days.
NOTE:Do not freeze the pool plate.
OBMRASIVD.R2
Total
volume
14
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Table 7. EmulsiFIRE Required Volumes
4 Samples + Controls
EWMX_1 EWMX_2 EWMX_3 EWMX_4 EWMX_5 EWMX_6 EWMX_7
EWMX 1 rxn (KR2)
(KR3) (KR4A) (KR4B) (NR2) (NR3) (NR4)
1
14
14
7
7
14
14
14
EM 1-7
(µL)
13.2
184.8
184.8
92.4
92.4
184.8
184.8
184.8
Beads
(µL)
0.8
11.2
11.2
5.6
5.6
11.2
11.2
11.2
EmEnz
(µL)
1.0
14.0
14.0
7.0
7.0
14.0
14.0
14.0
Sum
(µL):
15.0
210.0
210.0
105.0
105.0
210.0
210.0
210.0
210
210
105
105
210
210
210
no of rxn
Total
volume
Sample Batch Size (Including controls)
EmulsiFIRE
1
16
16
8
8
16
16
16
13.2
211.2
211.2
105.6
105.6
211.2
211.2
211.2
Beads
(µL)
0.8
12.8
12.8
6.4
6.4
12.8
12.8
12.8
EmEnz
(µL)
1.0
16.0
16.0
8.0
8.0
16.0
16.0
16.0
Sum
(µL):
15.0
240.0
240.0
120.0
120.0
240.0
240.0
240.0
240
240
120
120
240
240
240
Total
volume
3
4
5
6
4.5 mL
5.5 mL
6 mL
7 mL
8 mL
Figure 3. Using the electronic 12-channel pipette Program E
(refer to Table 5), take 70 µL EmulsiFIRE from the reagent
reservoir, dispense into the emulsion PCR plate beginning
in the row circled in the figure and mix 30 times per well.
Continue to dispense down the plate. During mixing, the
pipette will automatically aspirate the set volume, mix it in
the tip, and then dispense the set volume back into the wells.
5 Samples + Controls
EM 1-7
(µL)
2
3.5 mL
92. Position the emulsion PCR plate vertically as shown in
EWMX_1 EWMX_2 EWMX_3 EWMX_4 EWMX_5 EWMX_6 EWMX_7
EWMX 1 rxn (KR2)
(KR3) (KR4A) (KR4B) (NR2) (NR3) (NR4)
no of rxn
1
Figure 3. PCR Plate Positioning
6 Samples + Controls
EWMX_1 EWMX_2 EWMX_3 EWMX_4 EWMX_5 EWMX_6 EWMX_7
EWMX 1 rxn (KR2)
(KR3) (KR4A) (KR4B) (NR2) (NR3) (NR4)
1
18
18
9
9
18
18
18
EM 1-7
(µL)
13.2
237.6
237.6
118.8
118.8
237.6
237.6
237.6
Beads
(µL)
0.8
14.4
14.4
7.2
7.2
14.4
14.4
14.4
EmEnz
(µL)
1.0
18.0
18.0
9.0
9.0
18.0
18.0
18.0
Sum
(µL):
15.0
270.0
270.0
135.0
135.0
270.0
270.0
270.0
270
270
135
135
270
270
270
no of rxn
Total
volume
NOTE: To enhance the mixing process, keep moving your
hand while moving the pipette slowly up and down.
During aspiration, slowly lower the pipette into
the well, and while dispensing, lift the pipette up.
Be careful and avoid lifting the pipette tip out of
the fluid. To prevent cross-contamination of liquid
these movements have to be made slowly.
93. Visually inspect all samples for proper mixing. Phase
separation or precipitation of beads should not be present.
NOTE:Emulsion PCR wells which show phase separation or
precipitated beads after emulsion mixing need to be
repeated beginning from the Emulsion PCR section.
94. Seal the emulsion plate with transparent adhesive PCR
film. Ensure all wells are entirely covered by foil and foil is
not damaged.
95. Place the emulsion plate in a thermal cycler within
15 minutes.
96. Close the cycler lid, select and run the
“Emulsion PCR” program.
NOTE:Emulsion PCR program is set up by
Applications Specialist.
97. Empty the remaining EmulsiFIRE into a conical tube and
discard conical tube and reservoir into waste container.
CAUTION:The oil in EmulsiFIRE dissolves plastic! Therefore it
should not be stored in the reservoir.
98. After the run, print and/or save report. Carefully remove
emulsion PCR plate from the cycler.
99. Check the emulsion plate wells after cycling and record any
deviations (listed below). Document which wells are affected.
• Different liquid levels among the wells
• No phase separation
• Precipitated beads
NOTE: Phase separation should be present. It must be seen
as a white layer on top and a brown layer on bottom
as shown in Figure 4.
a: EWMX = Emulsion Working Mixes, EM = Emulsion Master Mix,
EmEnz = Emulsion PCR DNA Polymerase, rxn = reactions, KR = KRAS Exon,
NR = NRAS Exon
Emulsion PCR
88. Using a multi-channel pipette, add 15 µL per well of each
EWMX into the emulsion plate. Use Emulsion PCR work order
template to ensure that each EWMX gets transferred into the
right well positions of the emulsion plate.
89. Shake EmulsiFIRE vigorously by hand for 30 seconds
± 5 seconds (do not vortex) and let settle (at least 5 minutes
but no longer than 30 minutes) until no air bubbles remain.
NOTE:Check time with stopwatch.
90. Add 5 µL of template (samples and controls) from the
final dilution plate into each corresponding well of the
emulsion plate and mix by pipetting up and down 5 times.
Use Emulsion PCR work order template to ensure that each
sample and control is dispensed into the right well positions
of the emulsion plate.
CAUTION:Visually check if liquid levels in all pipette tips are
the same.
91. Transfer required volume of EmulsiFIRE into a disposable
reagent reservoir according to Table 7.
CAUTION:Handle EmulsiFIRE with caution. It contains bis
(2-ethylhexyl) carbonate which causes skin irritation.
OBMRASIVD.R2
15
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NOTE: Emulsion PCR wells, which show no phase separation,
reduced liquid levels, or precipitated beads after
emulsion cycling, need to be repeated. Start from the
Emulsion PCR section.
Preparation of Hybridization Buffer-Probe Mixes
106.Transfer the required volume of Hybridization Buffer into a 15
or 50 mL tube. Refer to Table 10.
CAUTION: Handle Hybridization Buffer with caution. It contains
Tetramethylammonium chloride (TMAC), which
causes skin, eye, and respiratory irritation. Wear
laboratory coat, powder-free gloves and protective
eyeglasses when handling Hybridization Buffer and
Hybridization Buffer-Probe Mixes.
Figure 4. Emulsion PCR Phase Separation
Table 10. Hybridization Buffer Required Volumes
Sample Batch Size (Including controls)
Hybridization Buffer
100.Store the emulsion PCR plate at 2 to 8°C for a maximum of
3 days.
2
3
4
5
6
13 mL
17 mL
21 mL
25 mL
30 mL
34 mL
8 mL
1
2
3
4
5
6
328
328
394
460
525
591
5
5
6
7
8
9
333
333
400
467
533
600
Hybridization Probe
Mixes (1-12) (µL)
Total volume (µL)
109.Mix Hybridization Buffer-Probe Mixes by pipetting up and
down for at least 15 times and then close tube strips.
110.Store Hybridization Buffer-Probe Mixes in the dark at room
temperature for a maximum of 3 hours.
Breaking and Hybridization
NOTE:During the entire breaking and hybridization process,
take care that there are no air bubbles at the bottom
of the wells. This could lead to insufficient breaking
and hybridization process and/or loss of beads due to
insufficient magnet recovery.
NOTE:Check incubation times using stopwatch.
111.Transfer required volume of Breaking Buffer 1 (BB1) into a
reagent reservoir, according to Table 12.
entered into the electronic pipettor, according to Table 9, and
check that battery power is at least 50%:
Table 9. Electronic Pipettor Specifications
(Abbreviations defined)a
Table 12. Breaking Buffer 1 Required Volumes
Program Name
B
H
P
100
70
80
4
4
4
100
70
80
Dispense speed
4
4
4
Mixing cycles
20
20
15
Mixing speed
8
8
8
Sample Batch Size (Including controls)
BB1
1
2
3
4
5
6
9 mL
12 mL
14 mL
16 mL
19 mL
21 mL
112.Place the emulsion PCR plate in a rack and carefully remove
the adhesive transparent film.
113.Add 100 µL of BB1 to each well and mix by pipetting up
and down using electronic pipette Program B, according to
Table 9. The solution must be homogenous and no magnetic
bead aggregates should be visible immediately after mixing.
Continue pipetting up and down in case this is not fulfilled.
a: B= Breaking, H= Hybridization, P= Phosphate Buffered Saline (PBS) Wash
NOTE:Programs are set up by Applications Specialist.
OBMRASIVD.R2
6
7.5 mL
Hybridization Buffer (µL)
105.Ensure the following electronic pipette programs have been
Dispense volume (µL)
5
7 mL
Sample Batch Size (Including controls)
Sample Batch Size (Including controls)
1
Aspirate speed
4
6 mL
Table 11. Preparation of Hybridization Buffer-Probe
Mixes
Table 8. PBS Required Volumes
Aspirate volume (µL)
3
5 mL
light protecting box. Vortex each probe mix vial for 5 seconds
and spin down with centrifuge for 2 seconds. Label tubes of
provided tube strips with “HPMx 1 to 12”.
108.Prepare Hybridization Buffer-Probe Mixes (12 probe mixes)
by pipetting the required volume of Hybridization Buffer
first and then pipetting the required volume of Hybridization
Probe Mix into labeled tube strips, according to Table 11.
Ensure that the correct HPMx vial is used to prepare each
corresponding probe mix.
NOTE:Do not pipette volumes < 5 µL. Discard pipette tips in
TMAC solid waste container.
Use Breaking and Hybridization work order template to record
relevant information:
Preparation
When using new breaking buffers, prepare the solutions as
described below.
101.Prepare Breaking Buffer 1 (BB1) solution when opening new
reagent kit. Before use, add 80 mL of 2-Butanol and 70 mL of
1-Propanol to the BB1 concentrate, to obtain 200 mL of BB1
working solution. Mix bottle by inverting 10 times. Store at
15 to 25°C.
102.Prepare Breaking Buffer 2 (BB2) solution when opening new
reagent kit. Before use, add 10 mL of 2-Butanol and 10 mL of
1-Propanol to the BB2 concentrate, to obtain 100 mL of BB2
working solution. Mix bottle by inverting 10 times.
Store at 15 to 25°C.
103.If emulsion PCR plate was stored after run at 2 to 8°C,
re‑check the wells as described in step 99 of the
Emulsion PCR section. This step is not necessary if emulsion
plate is processed immediately after the emulsion PCR run.
104.Transfer required PBS amount into a 50 mL conical tube.
Refer to Table 8. Bring PBS to room temperature prior to use
in washing steps.
Specification
2
5 mL
107.Remove Hybridization Probe Mixes (HPMx 1 to 12) from
Breaking and Hybridization
PBS
1
16
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NOTE:When mixing, the pipette tips must reach the
bottom of the wells. If the solution has not become
homogenized immediately after mixing, i.e. phase
separation is still visible, repeat pipetting up and
down until solution is homogenous. As time elapses,
it is normal to observe phase separation in the wells.
114.Incubate the plate for 1 minute at room temperature
(15 to 25°C).
115.Place the 96-well PCR plate on the 96-well magnet for 1 minute.
116.Adjust manual pipette to 200 µL volume and completely
remove supernatant. Carefully dip the pipette tip in the
middle of the well without contacting the walls.
117.Remove the 96-well PCR plate from the 96-well magnet and
place in the rack.
118.Pipette 100 µL of BB1 into every well and mix by pipetting up
and down at least 20 times at the bottom of the well using
electronic pipette Program B, according to Table 9.
119.Visually inspect PCR plate to ensure that the beads are
thoroughly resuspended. If magnetic bead aggregates are
visible, continue pipetting up and down in the affected wells.
120.Place the 96-well PCR plate on the 96-well magnet for
2.5 minutes.
121.Transfer required volume of Breaking Buffer 2 (BB2) into a
reagent reservoir, according to Table 13.
133.Place the 96-well PCR plate in the PCR machine, close the
cycler lid, and start Hybridization program within 15 minutes.
NOTE:Hybridization program is set up by
Applications Specialist.
134.After the run, print and/or save report, and carefully remove
PCR plate from the cycler.
Washing
NOTE:Execute the washing procedure within 15 minutes of
Hybridization program completion. Use protective
eyeglasses throughout steps 136 to 138.
135.Transfer PBS equilibrated to room temperature from 50 mL
conical tube into a reagent reservoir.
NOTE:Maximum volume capacity of reagent reservoir is
25 mL.
136.Take the plate in a 96-well PCR plate rack and carefully peel
the film.
137.Place the 96-well PCR plate on the 96-well magnet for
1 minute.
138.Adjust manual pipette to 100 µL volume and completely
remove supernatant. Carefully dip the pipette tip in the
middle of the well without contacting the walls.
NOTE:Discard supernatant in TMAC liquid waste and pipette
tips in TMAC solid waste.
139.Remove the 96-well PCR plate from the 96-well magnet and
place in rack.
140.Pipet 80 µL of PBS into each well and mix by pipetting up
and down at least 15 times at the bottom of the well using
electronic pipette Program P, according to Table 9.
141.Visually inspect PCR plate to ensure that the beads are
thoroughly resuspended. If magnetic bead aggregates are
visible, continue pipetting up and down in the affected wells.
142.Place the 96-well PCR plate on the 96-well magnet for
1 minute.
143.Adjust manual pipette to 60 µL volume and remove
supernatant. Carefully dip the pipette tip in the middle of the
well without contacting the walls.
144.Remove 96-well PCR plate from the 96-well magnet and place
in rack.
145.Pipet 80 µL of PBS into each well and mix by pipetting up
and down at least 15 times at the bottom of the well using
electronic pipette Program P, according to Table 9.
146.Visually inspect PCR plate to ensure that the beads are
thoroughly resuspended. If magnetic bead aggregates are
visible, continue pipetting up and down in the affected wells.
147.Place the 96-well PCR plate on the 96-well magnet for
1 minute.
148.Adjust manual pipette to 80 µL volume and remove
supernatant. Carefully dip the pipette tip in the middle of the
well without contacting the walls.
149.Remove the 96-well PCR plate from the 96-well magnet and
place in the rack.
150.Pipet 80 µL of PBS into each well and mix by pipetting up
and down at least 15 times at the bottom of the well using
electronic pipette Program P, according to Table 9.
151.Visually inspect PCR plate to ensure that the beads are
thoroughly resuspended. If magnetic bead aggregates are
visible, continue pipetting up and down in the affected wells.
152.Pipet again 80 µL of PBS into each well and mix by pipetting
up and down at least 15 times at the bottom of the well using
electronic pipette Program P, according to Table 9.
Table 13. Breaking Buffer 2 Required Volumes
Sample Batch Size (Including controls)
BB2
1
2
3
4
5
6
5 mL
6 mL
7 mL
8 mL
10 mL
11 mL
122.Adjust manual pipette to 140 µL volume and remove
supernatant. Carefully dip the pipette tip in the middle of the
well without contacting the walls.
123.Remove the 96-well PCR plate from the 96-well magnet and
place in the rack.
124.Pipet 100 µL of BB2 into every well and mix by pipetting up
and down at least 20 times at the bottom of the well using
electronic pipette Program B, according to Table 9.
125.Visually inspect PCR plate to ensure that the beads are
thoroughly resuspended. If magnetic bead aggregates are
visible, continue pipetting up and down in the affected wells.
126.Incubate the 96-well PCR plate at room temperature
(15 to 25°C) for 2 minutes.
127.Place the 96-well PCR plate on the 96-well magnet for 1 minute.
128.Adjust manual pipette to 140 µL volume and remove
supernatant. Carefully dip the pipette tip in the middle of the
well without contacting the walls.
129.Remove 96-well PCR plate from the 96-well magnet and place
in a rack.
130.Pipet 70 µL of each corresponding Hybridization BufferProbe Mix into every well of the corresponding PCR plate
column and mix by pipetting up and down at least 20 times
at the bottom of the well using electronic pipette Program H,
according to Table 9.
NOTE:Use protective eyeglasses during the process. Discard
pipette tips in TMAC solid waste.
131.Visually inspect PCR plate to ensure that the beads are
thoroughly resuspended. If magnetic bead aggregates are
visible, continue pipetting up and down in the affected wells.
132.Seal the 96-well PCR plate with transparent adhesive foil.
Ensure by visual inspection that wells are entirely covered by
the foil and foil is not damaged.
OBMRASIVD.R2
17
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153.Visually inspect PCR plate to ensure that the beads are
Table 14. Specific Analysis Template
thoroughly resuspended. If magnetic bead aggregates are
visible, continue pipetting up and down in the affected wells.
154.Proceed to flow cytometry analysis. If the plate cannot be
analyzed in the flow cytometer within 30 minutes, it must
be shielded from light, stored at 2 to 8°C in the post-PCR
laboratory and analyzed not later than 3.5 days. Samples
and controls must be resuspended again immediately before
placing the plate in the flow cytometer following instructions
in the next section.
Gene/Exon/ Well Positions/
Codon
Columns
Data Acquisition with Flow Cytometer
Preparation
155.Generate Flow worklist using the flow cytometer
worklist template.
156.Power up the Cube 6i flow cytometer.
157.Refer to the Cube 6i manual for instructions on instrument
qualification (QC). Document that the instrument has been
qualified for running the OncoBEAM assay before use.
NOTE: Step 158 (Resuspension) is not necessary if the plate
is used < 30 minutes after hybridization.
158.Place the plate in a plate rack and carefully remove the top
foil from the plate. Position the pipette tips towards the
bottom of each well to ensure proper mixing by pipetting up
and down at least 15 times at the bottom of the well using
electronic pipette Program P, according to Table 9.
Ensure by visual inspection that the beads are thoroughly
resuspended and that no air bubbles are visible at the bottom
of the wells.
CAUTION:Danger of contamination. Be careful not to carryover
material from the wells.
Measurement Using Cube 6i Flow Cytometer
159.Select Acquisition template for OncoBEAM assay.
160.Place the 96-well plate in the autoloader, load the worklist
and perform measurement as described in the Cube 6i flow
cytometer manual.
NOTE:Reliable measurement is only guaranteed if 96-well
plate is placed in correct orientation with the required
adapter plate. Ensure that the plate position for
measurement in CyView matches the plate position
on the autoloader. Check that the processing order
direction setting in CyView is “top-down” to ensure
that the worklist is imported in the correct order, that
samples/controls are measured in the correct order,
and that there is correct linkage for sample/control
positions and measurements. Non conformity results
in incorrect linkage of sample/control positions and
measurements. Ensure foil is removed from the PCR
plate before measurement. Non conformity results in
no measurement and/or damage of the device.
161.After the run, seal the plate and store at 2 to 8°C in the dark
for up to 3.5 days for potential rerun. Print Run Conditions
Report and proceed with data analysis.
Comment
A-H 1
KR2Cdn12_KR2Cdn13_RAS_CRC.fey
KR2_Cdn13
A-H 2
KR2Cdn12_KR2Cdn13_RAS_CRC.fey
Combined
template
KR3_Cdn59
A-H 3
KR3Cdn59_KR3Cdn61_RAS_CRC.fey
KR3_Cdn61
A-H 4
KR3Cdn59_KR3Cdn61_RAS_CRC.fey
KR4A_Cdn117
A-H 5
KR4B_Cdn146
A-H 6
NR2_Cdn12
A-H 7
NR2Cdn12_RAS_CRC.fey
Individual
template
NR2_Cdn13
A-H 8
NR2Cdn13_RAS_CRC.fey
Individual
template
Combined
template
Combined
template
KR4aCdn117_KR4bCdn146_RAS_CRC.fey Combined
KR4aCdn117_KR4bCdn146_RAS_CRC.fey template
NR3_Cdn59
A-H 9
NR3Cdn59_NR3Cdn61_RAS_CRC.fey
NR3_Cdn61
A-H 10
NR3Cdn59_NR3Cdn61_RAS_CRC.fey
NR4_Cdn117
A-H 11
NR4_Cdn146
A-H 12
NR4Cdn117_NR4Cdn146_RAS_CRC.fey Combined
NR4Cdn117_NR4Cdn146_RAS_CRC.fey template
164.Analysis template specific PDF summary sheets will
be generated. Automated data analysis provides a test
result for each sample and control on PDF summary sheets.
The following results are expected:
Table 15. Expected Results
Sample Class
Test Status
Next Step
Valid
Continue with Result Validation section
Positive
Control
Negative
Control
Sample
Invalid
See Retest section
Valid
Continue with Result Validation section
Invalid
See Retest section
Mutation Detected
Continue with Result Validation section
No Mutation Detected Continue with Result Validation section
Invalid
See Retest section
NOTE: In case no result for a sample or control is available due to
e.g. instrument error - see Rerun section to proceed.
No further test result interpretation is required. Overall sample
result is described in Result Validation section.
Mutant fraction as well as mutant bead count are reported for
informational purpose only.
Result Validation
165.Transfer results from Run Conditions Report and specific
summary sheets to result validation sheet to validate and
evaluate results.
166.Check the Run Conditions Report for run validity. Check
validity of the external run controls for each codon before
evaluating samples. In case an external control or sample test
fails, refer to Retesting Procedure section. If samples and
controls are valid, proceed to step 167.
167.The following overall sample results are expected (see
Table 16).
Table 16. Overall Sample Results
Sample Class
Sample
Interpretation of Results and Expected Values
Data Analysis with FCS Express (V5.0)
Overall Sample Result
Next Step
Mutation Detected
Result Report
No Mutation Detected
Result Report
No Result
No clear result, see Retest section for
assistance to continue
NOTE:For valid samples and results, Table 17 provides possible
assay result scenarios that could occur.
162.Open FCS express.
163.Run codon specific batch analysis with corresponding analysis
templates according to Table 14:
OBMRASIVD.R2
Analysis Templates
KR2_Cdn12
18
DocuSign Envelope ID: 825DA068-1DE5-471B-B6D1-989C098CA476
c. Only 1 codon specific control (PC and/or NC) fails
Table 17. Overall Result Determination Examples
Test
(highlighted gray) AND 1 or more test results are
Mutation Detected (D):
Perform retesting of the RESPECTIVE test including
respective controls for those samples in which no
mutation was detected (ND) in all of the other codon
tests (dashed line):
Result value combinations
Test (Codon x)
D
ND
ND
ND
IV
IV
IV
IV
IV
D
Test (Codon y) Test 1-12
D
D
ND
ND
D
IV
IV
ND
IV
IV
Test (Codon z)
D
D
D
ND
D
D
IV
ND
ND
ND
Overall result
D
D
D
ND
D
D
NR
NR
NR
D
D = Mutation Detected; ND = No Mutation Detected; IV = Invalid; NR = No Result
Test
T1
T2
T3
T4
T5
T6
T7
T8
T9
T10
T11
T12
Retesting Procedure
Well
1
2
3
4
5
6
7
8
9
10
11
12
NOTE: Contact OncoBEAM Support for assistance in
Retest evaluation.
168.Evaluate required retest procedure: If external run controls
or samples do not pass validation, following retest
scenarios apply:
Retest Scenarios
a. All external run controls fail (highlighted gray): Perform
retesting of ALL tests including controls for plate
(dashed line).
A
S1
ND
S1
ND
S1
ND
S1
ND
S1
ND
S1
ND
S1
ND
S1
ND
S1
ND
S1
ND
S1
ND
S1
ND
B
S2
ND
S2
ND
S2
ND
S2
ND
S2
ND
S2
ND
S2
D
S2
ND
S2
ND
S2
ND
S2
ND
S2
ND
C
S3
ND
S3
ND
S3
ND
S3
ND
S3
ND
S3
ND
S3
ND
S3
ND
S3
ND
S3
ND
S3
ND
S3
ND
D
S4
ND
S4
ND
S4
ND
S4
ND
S4
ND
S4
ND
S4
ND
S4
ND
S4
ND
S4
ND
S4
ND
S4
ND
E
S5
ND
S5
ND
S5
ND
S5
ND
S5
ND
S5
ND
S5
ND
S5
ND
S5
ND
S5
ND
S5
ND
S5
ND
F
S6
D
S6
ND
S6
ND
S6
ND
S6
ND
S6
ND
S6
ND
S6
ND
S6
ND
S6
ND
S6
ND
S6
ND
G
n
V
n
V
n
IV
n
V
n
V
n
V
n
V
n
V
n
V
n
V
n
V
n
V
H
p1
V
p1
V
p2
V
p2
V
p3
V
p4
V
p5
V
p5
V
p6
V
p6
V
p7
V
p7
V
Test
T1
T2
T3
T4
T5
T6
T7
T8
T9
T10
T11
T12
Well
1
2
3
4
5
6
7
8
9
10
11
12
A
S1
S1
S1
S1
S1
S1
S1
S1
S1
S1
S1
S1
B
S2
S2
S2
S2
S2
S2
S2
S2
S2
S2
S2
S2
C
S3
S3
S3
S3
S3
S3
S3
S3
S3
S3
S3
S3
D
S4
S4
S4
S4
S4
S4
S4
S4
S4
S4
S4
S4
E
S5
S5
S5
S5
S5
S5
S5
S5
S5
S5
S5
S5
F
S6
S6
S6
S6
S6
S6
S6
S6
S6
S6
S6
S6
G
n
IV
n
IV
n
IV
n
IV
n
IV
n
IV
n
IV
n
IV
n
IV
n
IV
n
IV
n
IV
H
p1
IV
p1
IV
p2
IV
p2
IV
p3
IV
p4
IV
p5
IV
p5
IV
p6
IV
p6
IV
p7
IV
p7
IV
S = Sample; n = No Template Control; p = Positive Control;
D = Mutation Detected; ND = No Mutation Detected; IV = Invalid; V = Valid
NOTE:Depending on the FAILED parameter (see Tables 19
through 21) a Rerun or a Repeat test is recommended.
Based on the results above, the samples for Test 3 have to
be retested because its controls were invalid. However, since
mutations were detected in Sample 2 [T7] and Sample 6 [T1]
they do not have to be retested because the overall results
for those samples can be reported as Mutation Detected (D).
d. If several controls fail (PC or NTC; highlighted gray),
contact OncoBEAM support for instructions on retesting
and troubleshooting.
S = Sample; n = No Template Control; p = Positive Control; IV = Invalid
b. Only 1 codon specific control (PC and/or NC) fails
(highlighted gray) AND all other test results are No
Mutation Detected (ND):
Perform retesting of the RESPECTIVE test including
respective controls for those samples in which no
mutation was detected (ND) in all of the other codon
tests (dashed line):
T1
Test
T3
T4
T5
T6
T7
T8
T9
T10
T11
T12
T1
T2
T3
T4
T5
T6
T7
T8
T9
T10
T11
T12
1
2
3
4
5
6
7
8
9
10
11
12
A
S1
S1
S1
S1
S1
S1
S1
S1
S1
S1
S1
S1
B
S2
S2
S2
S2
S2
S2
S2
S2
S2
S2
S2
S2
C
S3
S3
S3
S3
S3
S3
S3
S3
S3
S3
S3
S3
D
S4
S4
S4
S4
S4
S4
S4
S4
S4
S4
S4
S4
1
2
3
4
5
6
7
8
9
10
11
12
E
S5
S5
S5
S5
S5
S5
S5
S5
S5
S5
S5
S5
A
S1
ND
S1
ND
S1
S1
ND
S1
ND
S1
ND
S1
ND
S1
ND
S1
ND
S1
ND
S1
ND
S1
ND
F
S6
S6
S6
S6
S6
S6
S6
S6
S6
S6
S6
S6
B
S2
ND
S2
ND
S2
S2
ND
S2
ND
S2
ND
S2
ND
S2
ND
S2
ND
S2
ND
S2
ND
S2
ND
G
n
n
n
IV
n
n
n
IV
n
n
n
n
n
IV
n
C
S3
ND
S3
ND
S3
S3
ND
S3
ND
S3
ND
S3
ND
S3
ND
S3
ND
S3
ND
S3
ND
S3
ND
H
p5
p6
IV
p6
p7
p7
D
S4
ND
S4
ND
S4
S4
ND
S4
ND
S4
ND
S4
ND
S4
ND
S4
ND
S4
ND
S4
ND
S4
ND
E
S5
ND
S5
ND
S5
S5
ND
S5
ND
S5
ND
S5
ND
S5
ND
S5
ND
S5
ND
S5
ND
S5
ND
F
S6
ND
S6
ND
S6
S6
ND
S6
ND
S6
ND
S6
ND
S6
ND
S6
ND
S6
ND
S6
ND
S6
ND
G
n
V
n
V
n
IV
n
V
n
V
n
V
n
V
n
V
n
V
n
V
n
V
n
V
H
p1
V
p1
V
p2
V
p2
V
p3
V
p4
V
p5
V
p5
V
p6
V
p6
V
p7
V
p7
V
Well
T2
Test
Well
p1
p1
p2
p2
p3
p4
p5
S = Sample; n = No Template Control; p = Positive Control; IV = Invalid
e. Sample test fails [Measurement validity control (=MVC)
fails (highlighted gray)]: Perform retesting of the
RESPECTIVE test of the AFFECTED sample and the
respective controls (dashed line)
S = Sample; n = No Template Control; p = Positive Control;
ND = No Mutation Detected; IV = Invalid; V = Valid
Based on the results above, all the samples for Test 3 have
to be retested because its controls were invalid and all other
codon specific test results are No Mutation Detected (ND).
Test
T1
T2
T3
T4
T5
T6
T7
T8
T9
T10
T11
T12
Well
1
2
3
4
5
6
7
8
9
10
11
12
A
S1
S1
S1
S1
S1
S1
S1
S1
S1
S1
S1
S1
B
S2
S2
S2
S2
S2
S2
S2
S2
S2
S2
S2
S2
C
S3
S3
S3
S3
S3
S3
S3
S3
S3
S3
S3
S3
D
S4
S4
S4
S4
S4
S4
S4
S4
S4
S4
S4
S4
E
S5
S5
S5
S5
S5
S5
S5
S5
S5
S5
S5
S5
F
S6
S6
S6
S6
S6
S6
S6
S6
S6
S6
S6
S6
G
n
n
n
n
n
n
n
n
n
n
n
n
H
p1
p1
p2
p2
p3
p4
p5
p5
p6
p6
p7
p7
S = Sample; n = No Template Control; p = Positive Control
OBMRASIVD.R2
19
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169.Evaluate required next step and starting point of retesting
Table 19. Retesting Guide for Sample Test
procedure according to failed analysis parameter as described
in retesting guide Tables 19 through 21. Refer to Table 18 for
analysis parameter description.
NOTE:If a sample failed, always include related external
controls in retesting (rerun and repeat run – indicated
by (*) in Table 19).
If an external control failed, always execute retesting
for all samples of related codon. Exception: If
retesting procedure of an external control indicates a
rerun – here only the affected external controls needs
to be rerun (indicated by (**) in Tables 20 and 21).
Table 18. Analysis Parameter Description
Analysis Parameter (AP)
Abbreviation
Description
AP1
SB#
Single Bead Number
AP2
SBpos
Single Bead Position
AP3
IP1%
Gate 1 Plot 1 Percentage
Failure of Analysis Retest from
Parameter (AP)
section
Next step
AP1
EmPCR*
Go to
repeat test
section:
step 174
Contact support of Cube 6i if
problem occurs frequently.
AP2
Rerun*
Go to rerun
section:
step 170
If AP4 failed in addition it is
recommended to follow retest
description of AP4.
If AP3+AP4 failed in addition it
is recommended to rerun (go to
step 170).
AP3
Rerun*
Go to rerun
section:
step 170
N.A.
AP4: EB# < LL - low
bead count
EmPCR*
Go to
repeat test
section:
step 174
N.A.
AP4: EB# < LL low
DNA count only codons
from 1 exon
EmPCR*
Go to
repeat test
section:
step 174
N.A.
AP4: EB# < LL low
DNA count - Plasma/DNA
more than 1
Extraction*
exon is affected
Go to
repeat test
section:
step 176
N.A.
AP5: EB% > UL
EmPCR*
Go to
repeat test
section:
step 174
Repeat from EmPCR with
an extra 1:10 dilution in 1x
TE Buffer (low EDTA), when
preparing final dilution.
Rerun*
Go to rerun
section:
step 170
If AP4 failed in addition it is
recommended to follow retest
description of AP4.
If AP3+AP4 failed in addition it
is recommended to rerun (go to
step 170).
EmPCR*
Go to
repeat test
section:
step 174
N.A.
Rerun*
Go to rerun
section:
step 170
If AP4 failed in addition it is
recommended to follow retest
description of AP4.
If AP3+AP4 failed in addition it
is recommended to rerun (go to
step 170).
EmPCR*
Go to
repeat test
section:
step 174
N.A.
AP4
EB#
Extended Bead Number
AP5
EB%
Extended Bead Percentage
AP6
IP2%
Gate 1 Plot 2 Percentage
AP7
IIP2%
Gate 2 Plot 2 Percentage
AP8
IIIP2%
Gate 3 Plot 2 Percentage
AP9
EB%/IP2%
Extended Bead Percentage/Gate 1
Plot 2 Percentage = AP5/AP6
AP10
wtP3pos
Wild-type Beads Plot 3 Position
AP6
AP11
wtP3CV
Wild-type Beads Plot 3 Coefficient
of Variation
AP7
AP12
mtP3pos
Mutant Beads Plot 3 Position
AP8
AP13
mtP3CV
Mutant Beads Plot 3 Coefficient of
Variation
AP14
mtP3#
Mutant Beads Plot 3 Number
AP15
mtP3%
Mutant Plot 3 Percentage/Fraction
AP9
AP10: wtP3pos >
FL3 UL
AP11
AP12
AP13
AP14
AP15
OBMRASIVD.R2
20
Comment
N.A.
EmPCR = Emulsion PCR; LL = Lower Limit; UL = Upper Limit; FL3 = Fluorescence
Channel 3 for the Mutant Signal
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171.Transfer PBS equilibrated to room temperature from 50 mL
Table 20. Retesting Guide for External Positive Control (PC)
Failure of Analysis
Parameter (AP)
Retest from
section
Next step
EmPCR
Go to
repeat test
section:
step 174
Contact support of Cube 6i if
problem occurs frequently.
AP2
Rerun**
Go to rerun
section:
step 170
If AP4 failed in addition it is
recommended to follow retest
description of AP4.
If AP3+AP4 failed in addition it
is recommended to rerun (go to
step 170).
AP3
Rerun**
Go to rerun
section:
step 170
N.A.
EmPCR
Go to
repeat test
section:
step 174
Rerun**
If AP4 failed in addition it is
to follow retest
Go to rerun recommended
description of AP4.
section:
If AP3+AP4 failed in addition it
step 170
is recommended to rerun (go to
step 170).
EmPCR
Go to
repeat test
section:
step 174
N.A.
AP10: wtP3pos >
FL3 UL
Rerun**
Go to rerun
section:
step 170
If AP4 failed in addition it is
recommended to follow retest
description of AP4.
If AP3+AP4 failed in addition it
is recommended to rerun (go to
step 170).
AP11 through AP15
EmPCR
Go to
repeat test
section:
step 174
N.A.
AP1
AP4: EB# < LL - low
bead count
AP4: EB# < LL low
DNA count
AP5: EB% > UL
AP6
AP7
AP8
AP9
conical tube into a reagent reservoir.
172.Fill up the desired wells with 120 µl of PBS and resuspend
Comment
thoroughly. Ensure proper mixing by pipetting up and down
at least 15 times at the bottom of the well using electronic
pipette Program P, according to Table 9. Ensure by visual
inspection that the beads are thoroughly resuspended and
that no air bubbles are visible at the bottom of the wells.
173.Generate Flow worklist using the flow cytometer worklist
template for retesting by selecting only the affected wells and
proceed with step 156.
NOTE: For reruns add the suffix -RR to the Plate-ID.
NOTE: Only rerun wells of affected tests. For failed samples
also include related external controls in rerun. In case
external controls failed, only rerun failed controls.
If all codons fail, contact
OncoBEAM support for
assistance in Retest evaluation.
Repeat Test
A repeat test describes the retesting procedure when a new plate
for the same test is prepared – either starting from plasma/DNA
extraction or emulsion PCR.
NOTE: A Repeat test is recommended if retesting guide Tables 19
through 21 indicated this.
• If the determined starting point is Emulsion PCR go on with
the following procedure:
174.Check that pool plate was stored no longer than 7 days at
2 to 8°C and start repeat test with steps 73 to 75.
NOTE: If no valid pool plate is available defreeze mPCR plate
from –15 to –30°C storage and start repeat test with
steps 73 to 78.
175.Proceed with BEAMing procedure from step 79.
NOTE: For repeat test add the suffix -RT to the Plate-ID.
NOTE: For failed samples also include related external
controls in repeat test. In case external controls failed,
perform repeat test for all samples of affected codon.
• If the determined starting point is Plasma/DNA extraction go
on with the following procedure:
176.Check that plasma was stored no longer than 24 months at
≤ –70°C and start repeat test with step 12.
NOTE: If no valid plasma is available start repeat test with
new specimen collection. For repeat test add the
suffix -RT to the Plate-ID.
NOTE: For repeat test samples also include related external
controls in repeat test. In case external controls failed,
perform repeat test for all samples of affected codon.
EmPCR = Emulsion PCR; LL = Lower Limit; UL = Upper Limit; FL3 = Fluorescence
Channel 3 for the Mutant Signal
Table 21. Retesting Guide for No Template Control (NTC)
Failure of Analysis
Parameter (AP)
AP1
AP2
AP3
AP4: 1 NTC fails
AP4: >1 NTC fails
AP5 through AP13
Next step
EmPCR
Go to
repeat test
section:
step 174
Rerun**
Go to rerun
section:
step 170
N.A.
EmPCR
Go to
repeat test
section:
step 174
N.A.
Go to
Plasma/
repeat test
DNA
section:
1
extraction step 176
Comment
Contact support of Cube 6i if
problem occurs frequently.
Decontaminate lab environment
and repeat test using fresh
reagents.
Quality Control
Two types of external BEAMing controls are provided with the
kit - positive control(s) = PC and no template control = NTC.
The positive controls are specific for each exon of the KRAS
and NRAS genes, thus 7 different positive controls (low level
synthetic mutant DNA representing each of the target regions in
a wild‑type background) are included in each plate run. Tris EDTA
buffer without target DNA is used as a no template control and
included in each plate run. All controls are run in parallel with all
workflow steps from DNA amplification to result interpretation.
Each codon test is valid if the PC and NTC are valid. Contact
OncoBEAM Support if PC and NTC results are frequently invalid.
An internal control measurement is included into each sample
and external control measurement as a full process control.
For this purpose the confirmation of the presence of the target
amplicon within the human genome at an acceptable level is used,
ie. a region within each of the 7 target amplicons is detected
from the DNA of the sample or control. This region is outside of
the region interrogated by the mutation specific probe or the
wild‑type specific probe.
N.A.
AP14
See above (AP4)
AP15
N.A.
1
Retest from
section
Restart for samples from Plasma/DNA extraction – external controls are used
from mPCR.
EmPCR = Emulsion PCR; NTC = No Template Control
Rerun
A Rerun describes the retesting procedure when the initial plate
for the same test is refilled and measured again.
NOTE: Rerun is recommended in case of a loss of well
information (e.g. NO results due to wrong sample naming,
instrument error, software error) or if retesting guide
Tables 19 through 21 indicated this.
• If the determined starting point indicates a Rerun go on with
the following procedure:
170.Check if plate was stored in the dark no longer than 3.5 days
at 2°C to 8°C.
OBMRASIVD.R2
21
DocuSign Envelope ID: 825DA068-1DE5-471B-B6D1-989C098CA476
Limitations
Table 23. Verification of LoD of KRAS/NRAS Mutations
The test cannot differentiate between somatic and germline
mutations without the analysis of matched normal cell.
False negative and false positive results may occur for the
following reasons:
1. Incorrect handling of blood samples or plasma samples
(e.g. prolonged storage)
2. Rare polymorphisms within the region of interest
3. Heterogeneity of specimen
4. High mutant samples might cause false positive results in
neighboring samples. Use caution during workflow procedure
and reanalyze test results for confirmation in case of doubt.
The test is not recommended for use for patients undergoing
therapy, as low tumor burden may exist which may yield
inaccurate results.
BEAMing technology is a highly sensitive detection procedure
that uses polymerase chain reaction (PCR), therefore use caution
to avoid contamination of samples and reaction mixes by external
sources of DNA and/or PCR product in the test environment and
the DNA in the positive control.
Other mutations within the gene of interest or other genes are
not analyzed with this test.
Gene
KRAS 2
KRAS 3
KRAS 4A
KRAS 4B
Medium DNA
High DNA
11
43
KR3 cd61 a183c
7
11
44
KR4A cd117 a351c
6
15
43
KR4B cd146 g436a
6
12
37
NR2 cd12 g35a
6
10
39
NR3 cd61 c181a
7
13
45
NR4A cd117 g351t
7
12
43
g34a
100%
g34c
100%
g34c
100%
12
g34t
100%
g35a
100%
g35c
100%
g35t
100%
13
g38a
100%
59
g175a
100%
a182t
100%
61
117
146
a182g
100%
a183c
100%
a183t
100%
a351c
100%
Codon
Mutation
12
NRAS 2
13
59
NRAS 3
61
Hit Rate
g34t
100%
g35a
100%
g35c
100%
g35t
100%
g37c
100%
g38a
100%
g38t
100%
g175a
100%
c181a
100%
a182g
100%
a182t
100%
a351t
100%
a183c
100%
g436a
100%
a183t
100%
c437t
100%
g351c
100%
g351t
100%
g436a
100%
117
Diagnostic Accuracy
A study was conducted to demonstrate the ability of the
OncoBEAM RAS CRC assay to correctly discriminate RAS
mutation positive and RAS mutation negative clinical plasma
samples as compared to results obtained with reference methods
for tissue sample testing. The RAS status of specimens from
mCRC patients was determined using the following reference
methods on FFPE tumor tissue sections: allele-specific PCR
and sequencing (Sanger sequencing, pyrosequencing, and next
generation sequencing). Reference method testing was conducted
with CE-IVD approved kits and in-house validated methods.
Table 24 shows the agreement analysis between the OncoBEAM
RAS CRC assay and the reference methods for detection of RAS
mutations in a total of 238 samples from Stage IV CRC patients.
The diagnostic accuracy of OncoBEAM RAS CRC assay with
reference methods was evaluated by estimating the overall
percent agreement (OPA) for RAS mutation detection. The results
demonstrate an overall percent agreement (OPA) of 93.3%
(refer to Table 24).
Claimed LoD in a medium background DNA concentration was
confirmed by detecting 20 replicates with a probability of 95%
(including confidence interval) of the sample measurements as
“Mutation Detected” for 7 representative target analytes (KRAS
2 cd12 g35t, KRAS 2 cd161 a183c, KRAS 4A cd117 a351c, KRAS
4B cd146 g436a and NRAS 2 cd12 g35a, NRAS 3 cd61 c181a,
NRAS 4 cd117 g351t). For all 34 target mutations 3-fold LoD was
verified by detecting at least 5 replicates of 3-fold LoD samples
for each target analyte in a medium DNA background level with
a probability of 100% of the sample measurements as “Mutation
Detected” (refer to Table 23 for hit rate results).
Table 24. OncoBEAM RAS CRC Kit vs. Reference Methods
Concordance Results for Detection of RAS
Mutations in mCRC Specimens
Tissue RAS Result
OncoBEAM RAS CRC
Plasma RAS Result
Mutation Detected
No Mutation Detected
Total
OPA
OPA = Overall Percent Agreement
OBMRASIVD.R2
Gene
The clinical cutoff was transferred from tissue to plasma by
testing 99 clinical samples of known tissue RAS status with
plasma BEAMing.
A clinical cutoff (mutant bead count) was set for each codon in a
way to maximize the number of true results. Samples above the
cutoff with a defined specific mutation signal are determined as
“Mutation Detected”.
Samples below the cutoff or above the cutoff but with an
unspecific signal are determined as “No Mutation Detected”.
Table 22. Limit of Detection [ds copies/2 mL]
7
100%
Clinical Cutoff
Limit of Detection (LoD)
DNA extracted from plasma of healthy donors was adjusted
with wild-type genomic DNA to generate background DNA of 3
different levels: low DNA concentration (16.5 ng/2 mL plasma),
medium DNA concentration (77 ng/2 mL plasma), and high DNA
concentration (385 ng/2 mL plasma). Copies of synthetic double
strand DNA for KRAS and NRAS target analyte (refer to Table 21)
were spiked into each DNA background in serial dilutions. Eight
(8) replicates of each panel member were prepared and run using
1 lot of the OncoBEAM RAS CRC Kit. The LoD was calculated for
each tested target mutation from a probit regression model as the
analyte concentration at which, with a predefined probability of
95%, measurement results yield a “Mutation Detected” result, as
shown in Table 22.
Low DNA
Hit Rate
g34a
146
Analytical Sensitivity
KR2 cd12 g35t
Mutation
NRAS 4
Specific Performance Characteristics
Mutation
Codon
22
Mutation
Detected
No Mutation
Detected
Total
112
7
119
9
110
119
121
117
238
93.3% (222/238)
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16.Dressman D, Yan H, Traverso G, Kinzler KW, Vogelstein B.
Transforming single DNA molecules into fluorescent magnetic
particles for detection and enumeration of genetic variations.
Proc Natl Acad Sci USA. 2003 Jul 22;100(15):8817-22.
17.Diehl F, Schmidt K, Choti MA, et al. Circulating mutant DNA to
assess tumor dynamics. Nat Med. 2008 Sep;14(9):985-90.
18.Bettegowda C1, Sausen M, Leary RJ et al. Detection of
circulating tumor DNA in early- and late-stage human
malignancies. Sci Transl Med. 2014 Feb 19;6(224):224ra24.
19.Morelli MP, Overman MJ, Dasari A, et al. Characterizing the
patterns of clonal selection in circulating tumor DNA from
patients with colorectal cancer refractory to anti-EGFR
treatment. Ann Oncol. 2015 Apr;26(4):731-6.
20.Bokemeyer C, Köhne CH, Ciardiello F, et al. FOLFOX4 plus
cetuximab treatment and RAS mutations in colorectal cancer.
Eur J Cancer. 2015 Apr 30. pii: S0959-8049(15)00311-1.
21.Allegra CJ, Rumble RB, Hamilton SR, et al. Extended RAS
Gene Mutation Testing in Metastatic Colorectal Carcinoma to
Predict Response to Anti–Epidermal Growth Factor Receptor
Monoclonal Antibody Therapy: American Society of Clinical
Oncology Provisional Clinical Opinion Update 2015. Journal
of Clinical Oncology. 33, 1-9 (2015).
Bibliography
1. Allegra CJ, Jessup JM, Somerfield MR, et al. American Society
of Clinical Oncology provisional clinical opinion: testing for
KRAS gene mutations in patients with metastatic colorectal
carcinoma to predict response to anti-epidermal growth factor
receptor monoclonal antibody therapy. Journal of Clinical
Oncology: Official Journal of the American Society of Clinical
Oncology, 27(12), 2091–2096. (2009).
2. Benson AB, Venook AP, Bekaii-Saab T, et al. Colon cancer,
version 3.2014. JNCCN, 12(7), 1028–1059 (2014).
3. Sorich M, Wiese M, Rowland A, Kichenadasse G, et al;
Extended RAS mutations and anti-EGFR monoclonal antibody
survival benefit in metastatic colorectal cancer: a metaanalysis of randomized, controlled trials (2014) Annals of
Oncology 26: 13–21, (2015).
4. Di Fiore F, Blanchard F, Charbonnier F, et al. Clinical relevance
of KRAS mutation detection in metastatic colorectal cancer
treated by Cetuximab plus chemotherapy. British Journal of
Cancer, 96(8), 1166–1169. (2007).
5. Karapetis CS, Khambata-Ford S, Jonker DJ, et al. K-ras
mutations and benefit from cetuximab in advanced colorectal
cancer. The New England Journal of Medicine, 359(17),
1757–1765 (2008).
6. Tol J, Koopman M, Cats A, et al. Chemotherapy, bevacizumab,
and cetuximab in metastatic colorectal cancer. The New
England Journal of Medicine, 360(6), 563–572. (2009).
7. Bokemeyer C, Kohne CH, Ciardiello F, et al. Treatment
outcome according to tumor RAS mutation status in OPUS
study patients with metastatic colorectal cancer (mCRC)
randomized to FOLFOX4 with/without cetuximab. Journal of
Clinical Oncology, 32:5s(suppl; abstr 3505) (2014).
8. Ciardiello F, Lenz HJ, Kohne CH, et al. Treatment outcome
according to tumor RAS mutation status in CRYSTAL study
patients with metastatic colorectal cancer (mCRC) randomized
to FOLFIRI with/without cetuximab. Journal of Clinical
Oncology, 32:5s (suppl; abstr 3506) (2014).
9. Douillard JY, Oliner KS, Siena S, et al. Panitumumab-FOLFOX4
treatment and RAS mutations in colorectal cancer. The New
England Journal of Medicine, 369(11), 1023–1034. (2013).
10.Heinemann V, Von Weikersthal LF, Decker T. Randomized
comparison of FOLFIRI plus cetuximab versus FOLFIRI
plus bevacizumab as first-line treatment of KRAS-wild-type
metastatic colorectal cancer: German AIO study KRK-0306
(FIRE-3). In American Society of Clinical Oncology (Vol. 31,
p. 15). Journal of Clinical Oncology. (2013).
11.Peeters M, Oliner KS, Price TJ, et al. Analysis of KRAS/NRAS
mutations in phase 3 study 20050181 of panitumumab (pmab)
plus FOLFIRI versus FOLFIRI for second-line treatment (tx)
of metastatic colorectal cancer (mCRC). Journal of Clinical
Oncology, 32(suppl 3; abstr LBA387). (2014).
12.Tejpar, et al. Effect of KRAS and NRAS mutations on treatment
outcomes in patients with metastatic colorectal cancer
(mCRC) treated first-line with cetuximab plus FOLFOX4: New
results from the OPUS study. J Clin Oncol 32 (2014)
13.Engstrom PF, et al. NCCN Clinical Practice Guidelines in
Oncology™: Colon Cancer. National Comprehensive Cancer
Network. Version 3. (2010).
14.Van Cutsem, et al. Metastatic colorectal cancer: ESMO Clinical
Practice Guidelines. Annals of Oncology 25 (Supplement 3):
iii1–iii9, (2014).
15.Catalog of Somatic Mutations in Cancer, www.sanger.ac.uk/
genetics/CGP/cosmic.
OBMRASIVD.R2
Technical Assistance
For technical assistance, call your local Sysmex organization, or
visit the Sysmex Inostics Web site at
http://www.sysmex-inostics.com.
Copyrights, Trademarks, and Patents
This product may be covered by U.S. Patent Nos. 8048627 and
8715934, as well as other corresponding foreign patents.
Limited Use Label License:
Notice to Customer: Limited license
The purchase price of this Product includes a limited, nontransferable license under U.S. and foreign patents owned by
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purchaser by the purchase of this Product.
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Polymerase is manufactured by Thermo Fisher Scientific,
Inc. Thermo Scientific™ Phusion™ is trademark or registered
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© 2016 Sysmex Inostics
www.sysmex-inostics.com
June 2016
OBMRASIVD.R2
23