Instructions for Use

IGK Gene Clonality Assay

For identification of clonal immunoglobulin kappa light chain gene rearrangements
For RESEARCH USE ONLY. Not for use in diagnostic procedures.

Manufactured in U.S.A.

Storage Conditions: -85ºC to -65ºC(DNA controls may be separated from assay kits and stored at 2°C to 8°C)

Catalog #11020020
11020021
11020031

Products

Quantity

IGK Gene Clonality Assay for Gel Detection
IGK Gene Clonality Assay for ABI Fluorescence Detection
IGK Gene Clonality Assay MegaKit for ABI Fluorescence Detection
FOR RESEARCH USE ONLY; not for use in diagnostic procedures

33 Reactions
33 Reactions
330 Reactions

Page 2 of 18

Table of Contents
2.1.
2.2.

Background ........................................................................................................................................................................................ 3
Summary ........................................................................................................................................................................................... 3

3.1.
3.2.
3.3.

Polymerase Chain Reaction ............................................................................................................................................................... 3
Gel Detection ..................................................................................................................................................................................... 4
Differential Fluorescence Detection .................................................................................................................................................. 4

4.1.
4.2.
4.3.

Reagent Components ........................................................................................................................................................................ 5
Warnings and Precautions ................................................................................................................................................................. 6
Storage and Handling......................................................................................................................................................................... 6

5.1.
5.2.
5.3.
5.4.

Thermal cycler ................................................................................................................................................................................... 7
Electrophoresis Unit (for Gel Detection) ............................................................................................................................................ 7
UV Illumination Unit (for Gel Detection) ............................................................................................................................................ 7
ABI Capillary Electrophoresis Instruments (for ABI Detection) ........................................................................................................... 7

6.1.
6.2.
6.3.
6.4.
6.5.
6.6.

Precautions ........................................................................................................................................................................................ 8Interfering Substances ....................................................................................................................................................................... 8Specimen Requirements and Handling .............................................................................................................................................. 8
Sample Preparation ........................................................................................................................................................................... 8
Template Amplification Controls ....................................................................................................................................................... 8
Sample Storage .................................................................................................................................................................................. 8

7.1.
7.2.
7.3.
7.4.
7.5.
7.6.

Materials Provided............................................................................................................................................................................. 9
Materials Required (Not Provided) .................................................................................................................................................... 9Reagent Preparation ........................................................................................................................................................................ 10
Amplification.................................................................................................................................................................................... 11
Detection ......................................................................................................................................................................................... 11
Recommended Positive Controls ..................................................................................................................................................... 13

8.1.
8.2.

Analysis ............................................................................................................................................................................................ 13
Sample Interpretation...................................................................................................................................................................... 13

10.1.
10.2.

Expected Size of Amplified Products ................................................................................................................................................ 14
Sample Data..................................................................................................................................................................................... 14

14.1.
14.2.

Warranty and Liability...................................................................................................................................................................... 17
Patents and Trademarks .................................................................................................................................................................. 17

IGK Gene Clonality Assay

FOR RESEARCH USE ONLY; not for use in diagnostic procedures

280275 Rev. F | August 2023

Page 3 of 18

Assay Use
This Research Use Only assay identifies clonal immunoglobulin kappa light chain gene rearrangements and is useful for the
study of:

Identifying clonal B-cell populations
Evaluation of new research and methods in malignancy studies.

Summary and Explanation of the Test
2.1.

BackgroundRearrangements of the antigen receptor genes occur during ontogeny in B and T lymphocytes. These generearrangements generate products that are unique in length and sequence for each cell. Therefore, polymerase chain
reaction (PCR) assays can be used to identify lymphocyte populations derived from a single cell by detecting the uniqueV-J gene rearrangements present within these antigen receptor loci.1 This PCR assay employs multiple consensus DNA
primers that target conserved genetic regions within the immunoglobulin heavy chain gene. This test is used to detect
the vast majority of clonal B-cell malignancies from DNA. Test products can be analyzed using a variety of detection
formats, including gel and capillary electrophoresis.

2.2.

Summary
Invivoscribe’s gene clonality assays represent a simple approach to PCR-based clonality testing. These standardized
assays were carefully optimized testing positive and negative control samples using multiplex master mixes. This test
kit includes three (3) master mixes. The IGK Tube A master mix targets the variable (V) and the joining (J) regions of
the Ig kappa light chain locus. Whereas the IGK Tube B master mix targets kappa deleting element (Kde)rearrangements with the variable (V) region and the intragenic Jκ-Cκ region. The resulting Vκ-Kde and Jκ-Cκ intron-Kde
rearrangements are a result of unsuccessful rearrangements retained by the B-cell. The third master mix, theSpecimen Control Size Ladder, targets multiple genes and generates a series of amplicons of 96, 197, 297, 397 and 602
base pairs (bp) to ensure that the quality and quantity of input DNA is adequate to yield a valid result. A single thermal
cycler program and similar detection methodologies are used with all of our Gene Clonality Assays. This improves
consistency and facilitates cross training on a broad range of different assays.
This assay is based on the EuroClonality/BIOMED-2 Concerted Action
BMH4-CT98-3936.

Principles of the Procedure
3.1.

Polymerase Chain Reaction
Polymerase chain reaction (PCR) assays are routinely used for the identification of clonal B- & T-cell populations. This
test amplifies the DNA between primers that target the variable (V) and joining (J) regions (Tube A) or the variable,
intragenic and Kappa Deleting Element (Kde) regions (Tube B). These conserved regions lie on either side of an area
within the V-J region where programmed genetic rearrangements occur during maturation of all B and T lymphocytes.
The antigen receptor genes that undergo rearrangement are the immunoglobulin heavy chain and light chains genes in
B-cells and the T cell receptor genes in T-cells. Each B- and T-cell has a single productive V-J rearrangement that is
unique in both length and sequence. Therefore, when this region is amplified using DNA primers that flank this region,a clonal population of cells yields one or two prominent amplified products (amplicons) within the expected size range.
Two products are generated in cases when the initial rearrangement was non-productive and was followed byrearrangement of the other homologous chromosome. In contrast, DNA from a normal or polyclonal (many clones)
population produces a bell-shaped curve of amplicon products (or Gaussian distribution) that reflect theheterogeneous population of V-J region rearrangements.

IGK Gene Clonality Assay

280275 Rev. F | August 2023
FOR RESEARCH USE ONLY; not for use in diagnostic procedures

Page 4 of 18

Figure 1.

3.2.

Depicted is a simple representation of the organization of a rearranged immunoglobulin kappa lightchain gene on chromosome 2p11.2. Shown are relative positions and orientations for the Vκ, Jκ, andKde primers, which are included in the IGK master mix tubes.

Gel Detection
Gel electrophoresis, such as agarose gel electrophoresis or non-denaturing polyacrylamide gel electrophoresisis(PAGE), is commonly used to resolve the different amplicon products based on their size, charge, and conformation.
Since DNA is negatively charged, when an electrical potential (voltage) is applied across the gel containing PCR
products, the electrical field causes the amplicons to migrate through the gel. Smaller DNA fragments are able toeasily migrate through the gel matrix, whereas larger DNA fragments migrate more slowly. This causes a separation of
the amplicon products based on size. Ethidium bromide or other DNA intercalating dyes can then be used to stain anddetect these products in the gel.
A heteroduplex analysis can also be performed and run on a polyacrylamide gel to differentiate clonal and non-clonal
PCR products. A heteroduplex analysis involves denaturing the PCR products at a high temperature, then quickly reannealing the DNA strands by suddenly reducing the temperature. This causes a large portion of DNA strands toincorrectly bind to other non-homologous strands creating loops in the DNA. These loops cause a significant reductionin the ability of the DNA to migrate through a polyacrylamide gel. However, if the majority of the PCR products areclonal, when a heteroduplex analysis is performed, most of these PCR products will correctly re-anneal with ahomologous strand. These PCR products will run normally through the polyacrylamide gel. Therefore, in a clonal
sample with a polyclonal background, a heteroduplex analysis will cause most of the polyclonal product to run muchslower through the polyacrylamide gel, thereby increasing their separation and the ability to identify the clonalband(s).

3.3.

Differential Fluorescence Detection
Differential fluorescence detection is commonly used to resolve the different-sized amplicon products using a capillaryelectrophoresis instrument. Primers can be conjugated with several different fluorescent dyes (fluorophores) so thatthey can produce different emission spectra upon excitation by a laser in the capillary electrophoresis instrument. In
this manner, different fluorescent dyes can correspond to different targeted regions. This detection system results inunsurpassed sensitivity, single nucleotide resolution, differential product detection and relative quantification. In
addition, the use of agarose and polyacrylamide gels, as well as the use of carcinogens such as ethidium bromide, canvirtually be eliminated. Further, differential detection allows accurate, reproducible and objective interpretation ofprimer-specific products and automatic archiving of data. Inter-assay and intra-assay reproducibility in size
determination using capillary electrophoresis is approximately 1 to 2 nucleotides.

IGK Gene Clonality Assay

FOR RESEARCH USE ONLY; not for use in diagnostic procedures

280275 Rev. F | August 2023

Page 5 of 18

Reagents
4.1.

Reagent Components

Table 1. Available kits

Catalog #Description

Quantity

11020020

IGK Gene Clonality Assay – Gel Detection

33 Reactions

11020021

IGK Gene Clonality Assay – ABI Fluorescence Detection

33 Reactions

11020031

IGK Gene Clonality Assay MegaKit – ABI Fluorescence Detection

330 Reactions

Table 2. Reagent Components

Reagent

Catalog #Positive Control DNA

40880370

Negative (Normal)
Control DNA

40920010

Reagent Components(active ingredients)
IVS-0007 Clonal Control DNA

200 µg/mL of DNA in 1/10th TE

IVS-0000 Polyclonal Control DNA
200 µg/mL of DNA in 1/10th TE

Unit of
Quantity

Assay Kit
# of Units

MegaKit# of Units

100 µL1

5100 µL1

51500 µL

1

N/A

1500 µL

1

N/A

1500 µL

1

N/A

1500 µL

1

101500 µL

1

101500 µL

1

10

Storage Temp.

or

Gel Detection
IGK Tube A – Unlabeled21020010
Master Mixes

IGK Tube B – Unlabeled21020020

Amplification Control
Master Mix

Multiple oligonucleotides targeting the variable andjoining regions of the immunoglobulin kappa lightchain gene in a buffered salt solution.

20960020

Multiple oligonucleotides targeting the variable, Jκ Cκ intron, and Kde regions of the immunoglobulin
kappa light chain gene in a buffered salt solution.

Specimen Control Size Ladder – Unlabeled

Multiple oligonucleotides targeting housekeeping
genes.

ABI Fluorescence Detection
IGK Tube A – 6FAM
21020011
Master Mixes

IGK Tube B – 6FAM
21020021

Amplification Control
Master Mix
Note:

Multiple oligonucleotides targeting the variable andjoining regions of the immunoglobulin kappa lightchain gene in a buffered salt solution.

20960021

Multiple oligonucleotides targeting the variable, Jκ Cκ intron, and Kde regions of the immunoglobulin
kappa light chain gene in a buffered salt solution.

Specimen Control Size Ladder – 6FAM

Multiple oligonucleotides targeting housekeeping
genes.

There are no preservatives used in the manufacture of this kit.

IGK Gene Clonality Assay

280275 Rev. F | August 2023
FOR RESEARCH USE ONLY; not for use in diagnostic procedures

Page 6 of 18

4.2.

Warnings and Precautions

















4.3.

For RESEARCH USE ONLY. Not for use in diagnostic procedures.
Use this assay kit as a system. Do not substitute other manufacturer’s reagents. Dilution, reducing amplification
reaction volumes, or other deviation in this protocol may affect the performance of this test and/or nullify any
limited sublicense that comes with the purchase of this testing kit.
Materials are stable until the labeled expiration date when stored and handled as directed. Do not use kits beyond
their expiration date.
Close adherence to the protocol will assure optimal performance and reproducibility. Use care to ensure use ofcorrect thermal cycler program, as other programs may provide inaccurate/faulty data, such as false positive andfalse negative results.
Do not mix or combine reagents from kits with different lot numbers.
Wear appropriate personal protective equipment and follow good laboratory practices and universal precautions
when working with specimens. Handle specimens in approved biological safety containment facilities and open onlyin certified biological safety cabinets. Use molecular biology grade water for the preparation of specimen DNA.
Due to the analytical sensitivity of this test, use extreme care to avoid the contamination of reagents or amplification
mixtures with samples, controls or amplified materials. Closely monitor all reagents for signs of contamination (e.g.,
negative controls giving positive signals). Discard reagents suspected of contamination.
To minimize contamination, wear clean gloves when handling samples and reagents and routinely clean work areasand pipettes prior to doing PCR.
Autoclaving does not eliminate DNA contamination. Follow uni-directional workflow in the PCR laboratory; begin
with master mix preparation, move to specimen preparation, then to amplification, and finally to detection. Do notbring amplified DNA into the areas designated for master mix or specimen preparation.
Dedicate all pipettes, pipette tips, and any equipment used in a particular area to that area of the laboratory.
Use sterile, disposable plastic ware whenever possible to avoid RNase, DNase, or cross-contamination.

Storage and HandlingStore the assay at -85°C to -65°C until ready to use.
All reagents and controls must be thawed and vortexed or mixed thoroughly prior to use to ensure that they areresuspended completely.
Due to high salt concentrations, PCR master mixes are sensitive to freeze/thaw cycles. Minimize the exposure ofmaster mixes to freeze/thaw cycles.

If you have any questions, please contact the Invivoscribe technical staff. We would be happy to help you determine
your optimal storage needs.

IGK Gene Clonality Assay

FOR RESEARCH USE ONLY; not for use in diagnostic procedures

280275 Rev. F | August 2023

Page 7 of 18

Instruments
5.1.

Thermal cycler

Use or function: Amplification of DNA samples
Performance characteristics and specification:
o
o




5.2.

Follow manufacturer’s installation, operation, calibration, and maintenance procedures.
See section 7.4 Amplification for thermal cycler program.

Electrophoresis Unit (for Gel Detection)

Use or function: DNA fragment separation
Performance characteristics and specification:
o



5.3.

Capable of running at 35V to 135V for extended times

Follow manufacturer’s installation, operation, calibration, and maintenance procedures.

UV Illumination Unit (for Gel Detection)

Use or function: DNA detection
Performance characteristics and specification:
o



5.4.

Minimum Thermal Range: 15°C to 96°C
Minimum Ramping Speed: 0.8°C/sec

Capable of emitting light at a wavelength of ~302 nm

Follow manufacturer’s installation, operation, calibration, and maintenance procedures.

ABI Capillary Electrophoresis Instruments (for ABI Detection)

Use or function: Fragment detection and analysis
Performance characteristics and specification:o

The following capillary electrophoresis instruments will meet the performance needs for this assay:
ABI 310 Genetic Analyzer (1-capillary)
ABI 3100 Avant Genetic Analyzer (4-capillaries)
ABI 3100 Genetic Analyzer (16-capillaries)
ABI 3130 Genetic Analyzer (4-capillaries)
ABI 3130xL Genetic Analyzer (16-capillaries)
ABI 3500 Genetic Analyzer (8-capillaries)
ABI 3500xL Genetic Analyzer (24-capillaries)






Follow manufacturer’s installation, operation, calibration, and maintenance procedures.
The ABI instrument used must be calibrated with appropriate Matrix Standards as outlined in section 7.2 Materials
Required (not provided).
Use the default settings for your polymer and capillary type.
See section 7.5 Detection for more details.

IGK Gene Clonality Assay

280275 Rev. F | August 2023
FOR RESEARCH USE ONLY; not for use in diagnostic procedures

Page 8 of 18

Specimen Collection and Preparation
6.1.

Precautions
Biological specimens from humans may contain potentially infectious materials. Handle all specimens in accordancewith your institute’s Bloodborne Pathogen program and/or Biosafety Level 2 standards.

6.2.

Interfering Substances
The following substances are known to interfere with PCR:





6.3.

Divalent cation chelators
Low retention pipette tips
EDTA (not significant at low concentrations)
HeparinSpecimen Requirements and HandlingThis assay tests extracted and purified genomic DNA (gDNA). Common sources of gDNA include:



6.4.

Sample Preparation

6.4.1.
6.4.2.
6.4.3.

6.5.

5 cc of peripheral blood, bone marrow biopsy, or bone marrow aspirate anti-coagulated with heparin or EDTA; OR
Formalin-fixed paraffin embedded tissue or slides.

Test all samples in singlicate.
Test positive, negative and no template controls for each master mix.
Using any method of DNA extraction, extract the gDNA from unknown samples.
Resuspend DNA to final concentration of 100 µg to 400 µg per mL in 1/10th TE (1 mM Tris-HCl, pH 8.0; 0.1 mM
EDTA) or in molecular biology grade or USP water.
This is a robust assay system. A wide range of DNA concentrations will generate a valid result. Therefore,
quantifying and adjusting DNA concentrations is generally not necessary.
 When testing unknown samples, it is recommended to use a template amplification control master mix(e.g., Specimen Control Size Ladder) to verify the absence of PCR inhibitors and ensure sufficientquality and quantity of DNA is present to yield a valid result.

Template Amplification Controls
The Specimen Control Size Ladder master mix primers are labeled with a fluorescent dye (6-FAM). This label isdetected as BLUE using the differential fluorescence software. The amplicons produced with this master mix are at 96,
197, 297, 397 and 602 bp. Please note that the 96 bp band is comprised of 84 bp and 96 bp bands. Both of thesebands co-migrate on a gel. Run the products of this master mix separately.

6.6.

Sample Storage
Store samples using a method that prevents degradation of DNA.

IGK Gene Clonality Assay

FOR RESEARCH USE ONLY; not for use in diagnostic procedures

280275 Rev. F | August 2023

Page 9 of 18

Assay Procedure
7.1.

Materials Provided

Please see Table 2 for a list of provided materials.

7.2.

Materials Required (Not Provided)

Table 3. Materials Required (not provided)

Reagent/Material

DNA Polymerase

Recommended Reagents/Materials and Suppliers
Roche:• EagleTaq DNA Polymerase
Invivoscribe:• FalconTaq DNA Polymeraseor equivalent

Catalog
Number05206944190

Notes

N/A

60970130

Molecular Biology Grade or
USP Water

N/A

N/A

Sterile and free of DNase and RNase.

Calibrated Pipettes

N/A

N/A

Capable of accurately measuring
volumes between 1 µL and 1000 µL.

Thermal cycler

Thermo Fisher Scientific:• Veriti Dx Thermal Cycler
Bio-Rad:• MJ Research PTC-100 or PTC-200, PTC-220, PTC-240
Perkin-Elmer• PE 9600 or PE 9700

N/A

N/A

Vortex Mixer

N/A

N/A

N/A

PCR plates or tubes

N/A

N/A

SterileFilter barrier pipette tips

N/A

N/A

Sterile, RNase/DNase/Pyrogen-free

Microcentrifuge tubes

N/A

N/A

SterileN/A

For polyacrylamide gels

Gel Detection (for Agarose or Polyacrylamide Gels)
Gel Electrophoresis Unit

N/A
Thermo Fisher Scientific:• UltraPureTM 10 mg/mL Ethidium Bromide

15585-011

Agarose Gels

Thermo Fisher Scientific:• MetaPhorTM Agarose, 125 gor• LonzaTM NuSieveTM 3:1 Agarose

BMA50180
or
BMA50090

6% Polyacrylamide Gels

Thermo Fisher Scientific:• Novex TBE Gels (6%, 12 well)EC62652Box

TBE Running Buffer

Thermo Fisher Scientific:• Novex TBE Running Buffer (5X)

LC6675

Gel Loading Buffer

Thermo Fisher Scientific:• 10X BlueJuiceTM Gel Loading Buffer• Novex Hi-Density TBE Sample Buffer (5X)

10816-015
LC6678100 bp DNA Ladder

Thermo Fisher Scientific:• TrackItTM 100 bp DNA Ladder

10488-058

Ethidium Bromide

IGK Gene Clonality Assay

N/A

N/A

N/A
Dilute 1:5 prior to use.
N/A
N/A

280275 Rev. F | August 2023
FOR RESEARCH USE ONLY; not for use in diagnostic procedures

Page 10 of 18
Reagent/Material

Recommended Reagents/Materials and Suppliers

Catalog
Number

Notes

N/A

N/A

ABI Fluorescence Detection
ABI Capillary
Electrophoresis Instrument

Thermo Fisher Scientific:• ABI 310, 3100, 3130, or 3500 series

Hi-Di Formamide

Thermo Fisher Scientific:• Hi-DiTM Formamide

Size Standards

Invivoscribe:• Hi-Di Formamide w/ROX size standards for ABI 3100
Thermo Fisher Scientific:• For ABI 3100 or 3130 instruments:
o GeneScanTM - 400HD [ROX]TM• For ABI 3500 instruments:
o GeneScanTM - 600 [LIZ]TM v2.0

Spectral Calibration Dye
Sets

Polymer

Buffer

7.3.

Thermo Fisher Scientific:• For ABI 3100 and 3130 instruments:o DS-30 Matrix Standard Kit (Dye Set D)• For ABI 310 instruments:
o NED Matrix Standardo And Fluorescent Amidite Matrix Standards [6FAM,
TET, HEX, TAMRA, ROX]
• For ABI 3500 instruments:o DS-33 Matrix Standard Kit (Dye Set G5)

4311320N/A

60980061
402985

N/A44083994345827
402996401546

N/A

4345833

Thermo Fisher Scientific:• POP-4TM Polymer:o POP-4 for 310 Genetic Analyzerso POP-4 for 3100/3100-Avant Genetic Analyzerso POP-4 for 3130/3130xL Genetic Analyzers• POP-7TM Polymer:o POP-7 for 3130/3130xL Genetic Analyzerso POP-7 for 3500/3500xL Genetic Analyzers

43527594393714

Thermo Fisher Scientific:• 10X Genetic Analyzer Buffer with EDTA

40282440283843163554352755

N/A

Dilute 1:10 in sterile water before
use

Reagent Preparation
7.3.1.
7.3.2.

Using gloved hands, remove the master mixes from the freezer. Allow the tubes to thaw; then gently vortex tomix.
In containment hood or dead air box, aliquot the appropriate volume of each master mix into a sterile microfuge
tube (one tube for each master mix).


7.3.3.

Aliquot volumes are 45 μL for each sample + 135 μL (3 x 45 µL) for the positive, negative and no template(water) controls.
Include an additional 20 μL to correct for pipetting errors.

Add the appropriate amount of Taq DNA polymerase (0.25 μL @ 5 U/μL per 50 μL total PCR reaction volume) toeach master mix and gently mix by inverting several times or gently vortexing.


IGK Gene Clonality Assay

The master mixes are now ready for distribution to reaction tubes or plate, and addition of sample.

FOR RESEARCH USE ONLY; not for use in diagnostic procedures

280275 Rev. F | August 2023

Page 11 of 18

7.4.

Amplification
7.4.1.
7.4.2.
7.4.3.

Aliquot 45 μL of the master mix/enzyme solutions into individual PCR wells or tubes.
Add 5 μL of sample or control DNA to the individual tubes or wells containing the respective master mix
reactions. Pipette up and down several times to mix.
Amplify the reactions using the PCR program in Table 4.


Use the calculated option for temperature measurement with the PTC instruments.
Table 4. Thermal cycling conditions

Standard Program

7.4.4.

7.5.

Step

Temperature

Duration

Cycles1

95ᐤC

7 minutes

1

2

95ᐤC

45 seconds

3

60ᐤC

45 seconds

4

72ᐤC

90 seconds

5

72ᐤC

10 minutes

1

6

15ᐤC

∞

1

35

Remove the amplified PCR plate from the thermal cycler.

Detection


Not all detection formats are available for all assays.

Gel Detection – Agarose TBE Gels
7.5.1.
7.5.2.
7.5.3.
7.5.4.
7.5.5.

Prepare a 2% MetaPhor or NuSieve 3:1 agarose/TBE gel.
Individually mix 20 μL from each amplification reaction with 4 μL of 6X gel loading buffer.
Load 20 μL of this mixture into separate wells of the gel, flanked by DNA size standards.
Detect products using ethidium bromide or an equivalent dye.
Photograph the gel and interpret the data.

Gel Detection – Polyacrylamide TBE Gels
7.5.6.
7.5.7.
7.5.8.
7.5.9.

Assemble electrophoresis unit using a 6% non-denaturing polyacrylamide TBE gel (made with 1X TBE) and 0.5X
TBE running buffer.
Add 5 μL of ice-cold non-denaturing bromophenol blue loading buffer to samples.
Load 20 μL of mixture into wells of the gel.
Run gel at 110V for 2-3 hours or 40-50V overnight.


7.5.10.
7.5.11.
7.5.12.
7.5.13.

Voltage and electrophoresis time depend on the PCR amplicon size, acrylamide gel thickness, and type of
PCR equipment.
Voltage and run time can be adapted accordingly.

Stain gels in 0.5 µg/mL ethidium bromide (in water or 0.5X TBE Buffer) for 5-10 minutes.
Destain gels 2X in water for 5-10 minutes.
Visualize the gels with UV illumination.
Photograph the gel and interpret the data.

IGK Gene Clonality Assay

280275 Rev. F | August 2023
FOR RESEARCH USE ONLY; not for use in diagnostic procedures

Page 12 of 18

Gel Detection – Heteroduplex Analysis (RECOMMENDED)
7.5.14.
7.5.15.
7.5.16.
7.5.17.
7.5.18.
7.5.19.

Denature 20 μL of PCR products at 94ºC for 5 minutes.
Re-anneal PCR products at 4ºC for 60 minutes.
Assemble electrophoresis unit using a 6% non-denaturing polyacrylamide TBE gel (made with 1X TBE) and 0.5X
TBE running buffer.
Add 5 μL of ice-cold non-denaturing bromophenol blue loading buffer to samples
Load 20 μL of mixture into wells of the gel.
Run gel at 110V for 2-3 hours or 40-50V overnight.


7.5.20.
7.5.21.
7.5.22.
7.5.23.

Voltage and electrophoresis time depend on the PCR amplicon size, acrylamide gel thickness, and type of
PCR equipment.
Voltage and run time can be adapted accordingly.

Stain gels in 0.5µg/mL ethidium bromide (in water or 0.5X TBE Buffer) for 5-10 minutes.
Destain gels 2X in water for 5-10 minutes.
Visualize the gels with UV illumination.
Photograph the gel and interpret the data.

ABI Fluorescence Detection with ABI 310, 3100 & 3130 instruments
7.5.24.
7.5.25.
7.5.26.
7.5.27.
7.5.28.
7.5.29.
7.5.30.
7.5.31.

In a new microcentrifuge tube, mix an appropriate amount (10 μL per reaction) of Hi-Di Formamide with ROX
Size Standardsa. Vortex well.
In a new 96-well PCR plate, add 10 μL Hi-Di Formamide with ROX size standards to individual wells for eachreaction.
Transfer 1 μL of each reaction to the wells containing Hi-Di Formamide with ROX size standards. Add only one
sample per well. Pipette up and down to mix.
Cap or cover the PCR plate.
Heat denature the samples at 95 ºC for 2 minutes, then snap chill on ice for 5 minutes.
Prepare a sample sheet and injection list for the samples.
Run the samples on an ABI 310/3100/3130 capillary electrophoresis instrument according to its user manual.
Data are automatically displayed as size and color specific peaks. Review profile and controls.

ABI Fluorescence Detection with ABI 3500 instruments
Note:

7.5.32.
7.5.33.
7.5.34.
7.5.35.
7.5.36.
7.5.37.
7.5.38.
7.5.39.
aNote:

Due to instrument to instrument variation in the performance of the ABI 3500 platform, the amount offormamide, sample and size standard listed in the protocol is intended to be a starting point. The protocol may
need to be optimized for specific ABI 3500 Platforms.
In a new microcentrifuge tube, mix an appropriate amount (9.5 μL per reaction) of Hi-Di Formamide with LIZ
Size Standardsa. Vortex well.
In a new 96-well PCR plate, add 9.5 μL Hi-Di Formamide with LIZ size standards to individual wells for eachreaction.
Transfer 0.5 μL of each reaction to the wells containing Hi-Di Formamide with LIZ size standards. Add only one
sample per well. Pipette up and down to mix.
Cap or cover the PCR plate.
Heat denature the samples at 95ºC for 3 minutes, then snap chill on ice for 5 minutes.
Prepare a sample sheet and injection list for the samples.
Run the samples on an ABI 3500 capillary electrophoresis instrument according to its user manual.
Data are automatically displayed as size and color specific peaks. Review profile and controls.
Please see Applied Biosystems’ accompanying product insert for mixing Hi-Di Formamide with size standards fordifferent ABI instruments.

IGK Gene Clonality Assay

FOR RESEARCH USE ONLY; not for use in diagnostic procedures

280275 Rev. F | August 2023

Page 13 of 18

7.6.

Recommended Positive Controls

Table 5. Recommended positive controls and size of amplified products.

Master Mix

TargetColorControl DNA

Catalog #Product Size (bp)--40880370

120-160, 190-210, 260-300
143

IGK Tube A

Vκ-Jκ

Blue

Valid Size Range
IVS-0007 Clonal Control DNA

IGK Tube B

Vκ-Kde + intronKde

Blue

Valid Size Range
IVS-0007 Clonal Control DNA--40880370

210-250, 270-300, 350-390
274, 282

Specimen Control
Size LadderMultiple GenesBlue

Valid Size Range
IVS-0000 Polyclonal Control DNA--40920010

96, 197, 297, 397, 602
96, 197, 297, 397, 602

Note:

The amplicon sizes listed above were determined using an ABI platform. Amplicon sizes seen on your specific CE instrument may differ 1-4bp
from those listed above depending on the platform of detection (ABI) and the version of the analysis software used. Once identified, theamplicon size as determined on your specific platform will be consistent from run to run.

Interpretation and Reporting
This assay is for research use only; not intended for diagnostic purposes. PCR based testing does not identify 100% of clonalcell populations; therefore, repeat testing by Next Generation Sequencing (NGS) may be advisable to rule out clonality.

8.1.

Analysis
The size range for each master mix has been determined testing positive control samples. For accurate andmeaningful interpretation, ignore peaks that occur outside of the valid size range for each master mix. Peaks that areoutside of the range cannot be assumed to be valid.
Results can be reported as “positive” or “negative” for the “detection of clonal immunoglobin kappa light chain generearrangement consistent with the presence of a clonal cell population”.
8.1.1.
8.1.2.

Report samples that fail to amplify following repeat testing as “A result cannot be reported on this specimen
because there was DNA of insufficient quantity or quality for analysis”.
All assay controls must be examined prior to interpretation of sample results. If the controls do not yield thecorrect results, the assay is not valid and the samples cannot be interpreted.
The following describes the analysis for each control and the decisions necessary based upon the results:

Negative Control:o Positive:oPositive Control:
o
o



8.2.

Negative:

Positive:
Negative:(Polyclonal control, water or no template blank). If the negative control is:
Possible contamination of all PCR amplification reactions. Do not continue with
the interpretation of results. Prepare fresh master mix and repeat amplification.
Continue with the analysis.
(This can also be an extraction control if positive control material is taken throughextraction processes). If the positive control is:Continue with analysis.
Repeat the assay.

Specimen Control Size Ladder: (This is run on unknown samples only). If the Specimen Control Size Ladder:o Generates 96, 197, 297, 397 and 602 bp products: Continue with analysis. Smaller PCR fragments are
preferentially amplified, it is not unusual for the 602
bp fragment to have a diminished signal or to be
missing entirely.
o Does not generate the specified products:
Repeat assay and/or re-extract the specimen.

Sample Interpretation
Following the acceptance of the controls, the samples are interpreted as follows:
One or two prominent bands within the valid size range are reported as “Detection of clonal immunoglobulin kappalight chain gene rearrangement consistent with the presence of a clonal cell population.”

IGK Gene Clonality Assay

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FOR RESEARCH USE ONLY; not for use in diagnostic procedures

Page 14 of 18

Limitations of Procedure
The assay is subject to interference by degradation of DNA or inhibition of PCR due to heparin or other agents.

Expected Values
10.1.
Note:

Expected Size of Amplified Products“Color” indicates the color of products generated with the master mix when using the default color assignment
on ABI fluorescence detection systems.

Table 6. Expected Size of Amplified Products

Master Mix

TargetVk-Jk

IGK Tube A

Color

Control DNA

Catalog #Product Size in base pair (bp)Blue

Valid Size Range
IVS-0000 Polyclonal Control DNA
IVS-0007 Clonal Control DNA--40920010
40880370

120-160, 190-210, 260-300
135-155
143

IGK Tube B

Vk-KdeBlue

Valid Size Range
IVS-0000 Polyclonal Control DNA
IVS-0007 Clonal Control DNA--40920010
40880370

210-250, 270-300, 350-390
225-245, 265-285, 4041
274, 282

Specimen Control Size
LadderMultiple
Genes

Blue

Any Human DNA---

96, 197, 297, 397, 602
96, 197, 297, 397, 602

Note:
1Note:

10.2.

The amplicon sizes listed above were determined using an ABI platform. Amplicon sizes seen on your specific CE instrument may differ 1-4bp
from those listed above depending on the platform of detection (ABI) and the version of the analysis software used. Once identified, theamplicon size as determined on your specific platform will be consistent from run to run.
Under sub-optimal conditions a nonspecific product of 404 bp can be detected in Tube B. To discriminate between specific and nonspecific,
negative control DNA should not show this band within the same experiment. If a band is present, we then consider the band nonspecific.

Sample Data
Gel Detection. The data shown in Figures 2 and 3 were generated using the master mixes indicated. Lane 1 displays
data generated testing an alternative 100% clonal control DNA.

Figure 2. Amplified products using the IGK Tube A – Unlabeled
master mix were run on a 6% polyacrylamide gel.

IGK Gene Clonality Assay

Figure 3. Amplified products using the IGK Tube B – Unlabeled
master mix were run on a 6% polyacrylamide gel.

FOR RESEARCH USE ONLY; not for use in diagnostic procedures

280275 Rev. F | August 2023

Page 15 of 18

ABI Fluorescence Detection. The data shown in Figures 4 and 5 were generated using the master mixes indicated.
Amplified products were run on an ABI instrument.

Figure 4. Amplified products using the IGK Tube A – 6FAM master mix.

Figure 5. Amplified products using the IGK Tube B – 6FAM master mix.

IGK Gene Clonality Assay

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FOR RESEARCH USE ONLY; not for use in diagnostic procedures

Page 16 of 18

Technical and Customer Service
Thank you for purchasing our IGK Gene Clonality Assay. We appreciate your business. We are happy to assist you in thevalidation of this assay and will provide ongoing technical assistance to keep the assays performing efficiently in yourlaboratory. Technical assistance is most rapidly obtained using our Internet site: http://www.invivoscribe.com or by sending
an email inquiry to: support@invivoscribe.com.
Contact Information
Invivoscribe, Inc
10222 Barnes Canyon Road | Building 1 | San Diego | California 92121-2711 | USA
Phone: +1 858 224-6600 | Fax: +1 858 224-6601 | Business Hours: 7:00AM - 5:00 PM PST/PDT
Technical Service: support@invivoscribe.com | Customer Service: sales@invivoscribe.com | Website: www.invivoscribe.com

References
1.
2.
3.
4.

Rock, E.P., Sibbald, P.R., Davis, M.M. and Chien, Y. CDR3 Length in Antigen-specific Immune Receptors. J Exp Med. 1994 Jan
1;179(1):323-8.
Miller, JE, Wilson, SS, Jaye, DJ, Kronenberg, M. An automated semiquantitative B and T cell clonality assay. Mol. Diag. 1999,
4(2):101-117.
van Dongen, JJM et al. Design and standardization of PCR primers and protocols for detection of clonal immunoglobulin
and T-cell receptor gene recombinations in suspect lymphoproliferations: Report of the BIOMED-2 Concerted
Action BMH4-CT98-3936. Leukemia. 2003, 17(12):2257-2317.
van Krieken JH, Langerak AW, Macintyre EA, Kneba M, Hodges E, Sanz RG, Morgan GJ, Parreira A, Molina TJ, Cabeçadas J,
Gaulard P, Jasani B, Garcia JF, Ott M, Hannsmann ML, Berger F, Hummel M, Davi F, Brüggemann M, Lavender
FL, Schuuring E, Evans PA, White H, Salles G, Groenen PJ, Gameiro P, Pott Ch, van Dongen JJM. Improved
reliability of lymphoma diagnostics via PCR-based clonality testing: report of the BIOMED-2 Concerted Action
BHM4-CT98-3936. Leukemia. 2007; 21(2):201-6.

Symbols
The following symbols are used in labeling for Invivoscribe products.

IGK Gene Clonality Assay

Catalog Number

Expiration Date

Reagent Volume

Manufacturer

Lot Number

Consult Instructions for Use

Storage Conditions

Research Use Only

FOR RESEARCH USE ONLY; not for use in diagnostic procedures

280275 Rev. F | August 2023

Page 17 of 18

Legal Notice
14.1.

Warranty and Liability
Invivoscribe, Inc. (Invivoscribe®) is committed to providing the highest quality products. Invivoscribe® warrants that the
products meet or exceed the performance standards described in the Instructions For Use, as to products with such an
insert. If a product is covered by product specifications and does not perform as specified, our policy is to replace theproduct or credit the full purchase price. No other warranties of any kind, expressed or implied, are provided by
Invivoscribe®. Invivoscribe® liability shall not exceed the purchase price of the product. Invivoscribe shall have noliability for direct, indirect, consequential or incidental damages arising from the use, results of use, or inability to use
its products; product efficacy under purchaser controlled conditions in purchaser’s laboratory must be established andcontinually monitored through purchaser defined and controlled processes including but not limited to testing of
positive, negative, and blank controls every time a sample is tested. Ordering, acceptance, and use of productconstitutes purchaser acceptance of sole responsibility for assuring product efficacy and purchaser agreement to thelimitation of liability set forth in this paragraph.
This product is for Research Use Only; not for use in diagnostic procedures.

14.2.

Patents and Trademarks
This product is covered by one or more of the following: European Patent Number 1549764, European Patent Number
2418287, European Patent Number 2460889, Japanese Patent Number 4708029, United States Patent 8859748, andrelated pending and future applications. All of these patents and applications are licensed exclusively to
Invivoscribe®. Additional patents licensed to Invivoscribe covering some of these products apply elsewhere. Many ofthese products require nucleic acid amplification methods such as Polymerase Chain Reaction (PCR). No license underthese patents to use amplification processes or enzymes is conveyed expressly or by implication to the purchaser by
the purchase of this product.
©2023 Invivoscribe, Inc. All rights reserved. The trademarks mentioned herein are the property of Invivoscribe, Inc.
and/or its affiliates or (as to the trademarks of others used herein) their respective owners.

IGK Gene Clonality Assay

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Page 18 of 18

IGK Gene Clonality Assay: Single Page Guide
15.1.
15.2.

Using gloved hands, remove the master mixes from the freezer. Allow the tubes to thaw; then gently vortex to mix.
In a containment hood or dead air box, remove an appropriate aliquot to a sterile microfuge tube (one tube for eachmaster mix).
•
•

15.3.
15.4.
15.5.
15.6.

Aliquot volumes are 45 μL for each sample + 135 μL for the positive, negative and no template controls.
Include an additional 20 μL to correct for pipetting errors.

Add the appropriate amount of Taq DNA polymerase (0.25 μL @ 5 U/μL per 50 μL total PCR reaction volume) to eachmaster mix and gently mix by inverting several times or gently vortexing.
Aliquot 45 μL master mix to individual wells of a PCR plate.
Add 5 μL DNA from the unknown and control samples to individual tubes or wells containing the respective master mix
reactions and pipette up and down several times to mix.
Amplify target DNA using the universal thermal cycler program.

Gel Detection – Heteroduplex Analysis
15.7.
15.8.
15.9.
15.10.
15.11.
15.12.
15.13.
15.14.
15.15.
15.16.

Denature 20 μL of PCR products at 94ºC for 5 minutes.
Re-anneal PCR products at 4ºC for 60 minutes.
Assemble electrophoresis unit using a 6% non-denaturing polyacrylamide TBE gel and 0.5X TBE running buffer.
Add 5 μL of ice-cold non-denaturing bromophenol blue loading buffer to samples
Load 20 μL of mixture into wells of the gel.
Run gel at 110V for 2-3 hours or 40-50V overnight.
Stain gels in 0.5µg/mL ethidium bromide (in water or 0.5X TBE Buffer) for 5-10 minutes.
Destain gels 2X in water for 5-10 minutes.
Visualize gel using UV illumination.
Photograph gel and interpret the data.

ABI Fluorescence Detection with ABI 310, 3100 & 3130 instruments
15.17.
15.18.
15.19.
15.20.
15.21.
15.22.
15.23.
15.24.

In a new microcentrifuge tube, mix an appropriate amount (10 μL per reaction) of Hi-Di Formamide with ROX Size
Standards. Vortex well.
In a new 96-well PCR plate, add 10 µL Hi-Di Formamide with ROX size standards to individual wells for each reaction.
Transfer 1 μL of each reaction to the wells containing Hi-Di Formamide with ROX size standards. Add only one sample
per well. Pipette up and down to mix.
Cap or cover the PCR plate.
Heat denature the samples at 95 ºC for 2 minutes, then snap chill on ice for 5 minutes.
Prepare a sample sheet and injection list for the samples.
Run the samples on an ABI 310/3100/3130 capillary electrophoresis instrument according to the user manual.
Review profile and controls.

ABI Fluorescence Detection with ABI 3500 instruments
Note:

Due to instrument to instrument variation in the performance of the ABI 3500 platform, the amount of formamide,
sample and size standard listed in the protocol is intended to be a starting point. The protocol may need to be
optimized for specific ABI 3500 Platforms.

15.25.

In a new microcentrifuge tube, mix an appropriate amount (9.5 μL per reaction) of Hi-Di Formamide with LIZ Size
Standards. Vortex well.
In a new 96-well PCR plate, add 9.5 μL Hi-Di Formamide with LIZ size standards to individual wells for each reaction.
Transfer 0.5 μL of each reaction to the wells containing Hi-Di Formamide with LIZ size standards. Add only one sample
per well. Pipette up and down to mix.
Cap or cover the PCR plate.
Heat denature the samples at 95ºC for 3 minutes, then snap chill on ice for 5 minutes.
Prepare a sample sheet and injection list for the samples.
Run the samples on an ABI 3500 capillary electrophoresis instrument according to its user manual.
Review profile and controls.

15.26.
15.27.
15.28.
15.29.
15.30.
15.31.
15.32.

IGK Gene Clonality Assay

FOR RESEARCH USE ONLY; not for use in diagnostic procedures

280275 Rev. F | August 2023