Instructions for Use
T-Cell Receptor Gamma Gene Rearrangement Assay

For Identification of T-Cell Clonality.
This assay is 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#
12070051

Products

Quantity

T-Cell Receptor Gamma Gene Rearrangement Assay - ABI Fluorescence Detection
FOR RESEARCH USE ONLY; not for use in diagnostic procedures

30 Reactions

Page 2 of 17

TABLE OF CONTENTS
1.

ASSAY USE .......................................................................................................................................................................... 32.

SUMMARY AND EXPLANATION OF THE TEST ................................................................................................................................ 3
2.1.
Background.............................................................................................................................................................................. 3
2.2.
Summary ................................................................................................................................................................................. 3

3.

PRINCIPLES OF THE PROCEDURE ................................................................................................................................................ 3
Polymerase Chain Reaction (PCR) ........................................................................................................................................... 3
Differential Fluorescence Detection ........................................................................................................................................ 4

3.1.
3.2.

4.

REAGENTS ........................................................................................................................................................................... 5
4.1.
Reagent Components .............................................................................................................................................................. 5
4.2.
Warnings and Precautions....................................................................................................................................................... 6
4.3.
Storage and Handling .............................................................................................................................................................. 6

5.

INSTRUMENTS ...................................................................................................................................................................... 7
5.1.
Thermal cycler ......................................................................................................................................................................... 7
5.2.
ABI Capillary Electrophoresis Instruments .............................................................................................................................. 7

6.

SPECIMEN COLLECTION AND PREPARATION ................................................................................................................................. 8
Precautions.............................................................................................................................................................................. 8Interfering Substances............................................................................................................................................................. 8Specimen Requirements and Handling ................................................................................................................................... 8
Sample Preparation ................................................................................................................................................................. 8
Template Amplification Control .............................................................................................................................................. 8
Sample Storage........................................................................................................................................................................ 8

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

7.

ASSAY PROCEDURE ................................................................................................................................................................ 9
Materials Provided .................................................................................................................................................................. 9
Materials Required (not provided)............................................................................................................................................ 9Reagent Preparation ............................................................................................................................................................. 11
Amplification ......................................................................................................................................................................... 11
Detection ............................................................................................................................................................................... 11
Recommended Positive Controls........................................................................................................................................... 12

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

8.

INTERPRETATION AND REPORTING .......................................................................................................................................... 13
Sample Analysis ..................................................................................................................................................................... 13
Sample Interpretation ........................................................................................................................................................... 13

8.1.
8.2.

9.
10.

LIMITATIONS OF PROCEDURE ................................................................................................................................................. 13
EXPECTED RESULTS .............................................................................................................................................................. 14
Expected Size of Amplified Products ..................................................................................................................................... 14
Sample Data .......................................................................................................................................................................... 14

10.1.
10.2.

11.

TECHNICAL AND CUSTOMER SERVICE ....................................................................................................................................... 15

12.

REFERENCES ....................................................................................................................................................................... 1513.

SYMBOLS .......................................................................................................................................................................... 16

14.

LEGAL NOTICE .................................................................................................................................................................... 16
14.1.
Warranty and Liability ........................................................................................................................................................... 16
14.2.
Patents and Trademarks........................................................................................................................................................ 16

15.

T-CELL RECEPTOR GAMMA GENE REARRANGEMENT ASSAY: SINGLE PAGE GUIDE.............................................................................. 17

T-Cell Receptor Gamma Gene Rearrangement Assay

For RESEARCH USE ONLY; not intended for diagnostic use.

280280 Rev. G | September 2023

Page 3 of 17

1. Assay Use
This Research Use Only assay identifies T-Cell Receptor Gamma gene rearrangements and is useful for the study of clonal Tcell populations and evaluation of new research and methods in malignancy studies.

2. Summary and Explanation of the Test
2.1.

BackgroundRearrangement of the variable (Vγ) and joining (Jγ) gene segments of the TRG locus results in Vγ-Jγ products ofunique length and sequence. Clonal TRG rearrangements can be most rapidly identified by analyzing the size
distribution of DNA products amplified from conserved sequences that flank this Vγ-Jγ region.1 DNA isolated from anormal heterogeneous population of polyclonal T-cells produces a Gaussian distribution (bell-shaped size curve) of
amplified products. DNA amplified from a clonal T-cell population generates one or two product(s) of unique size thatreflects proliferation of a single rearranged clone.1,2
The T-cell receptor gamma (TRG, formerly known as TCRG) chain locus spans 160 kilobases on chromosome 7 (7p14).
The locus consists of 14 Vγ gene segments in six (6) subgroups, and five (5) Jγ gene segments interspersed between
two (2) constant (Cγ) gene segments. However, the repertoire of functional TRG molecules is limited to 4-6
functional Vγ gene segments that belong to two (2) subgroups.2

2.2.

Summary
Invivoscribe’s gene rearrangement and translocation assays represent a simple approach to PCR-based testing andutilize a series of standardized master mixes that have been carefully optimized. Several master mixes are used totest for rearrangements at each receptor locus and each master mix targets a different conserved region within thereceptor gene. This comprehensive testing approach maximizes identification of clonal rearrangements. A single
universal thermal cycler program and similar detection methodologies are used with all of our tests; this improves
consistency and facilitates cross-training.
This test kit includes three (3) master mixes. TCRG Mix 1 and TCRG Mix 2 target framework regions within thevariable (V) region and the joining (J) region of the TCR gamma chain locus. The Amplification Control master mix
targets the HLA-DQα locus and generates an amplicon of 235 base pairs (bp) to ensure the quality and quantity of
input DNA is adequate to yield a valid result.

3. Principles of the Procedure
3.1.

Polymerase Chain Reaction (PCR)
Polymerase chain reaction (PCR) assays are routinely used for the identification of clonal B- and T-cell populations.
These tests amplify the DNA between primers that target the conserved framework (FR) and joining (J) regions.
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 and light chains genes in B-cells and the T-cell receptor genes in Tcells. 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 incases when the initial rearrangement was non-productive and was followed by rearrangement of the otherhomologous chromosome. In contrast, DNA from a normal or polyclonal (many clones) population produces a bellshaped curve of amplicon products (or Gaussian distribution) that reflect the heterogeneous population of V-J region
rearrangements.
Since the antigen receptor genes are polymorphic (consisting of a heterogeneous population of related DNA
sequences), it is difficult to employ a single set of DNA primer sequences to target all of the conserved flanking
regions around the V-J rearrangement. N-region diversity and somatic mutation further scramble the DNA sequencesin these regions. Therefore, multiplex master mixes, which target several FR regions, are required to identify the
majority of clonal rearrangements. As indicated, clonal rearrangements are identified as prominent, single-sized

T-Cell Receptor Gamma Gene Rearrangement Assay

For RESEARCH USE ONLY; not intended for diagnostic use.

280280 Rev. G | September 2023

Page 4 of 17

products within the smear of different-sized amplicon products that form a Gaussian distribution around a
statistically favored, average-sized rearrangement. Please see Figure 1 for a diagram representing the organization ofa rearranged T-cell receptor gamma chain gene.

Figure 1.

3.2.

This is a diagrammatic representation of the T-cell receptor gamma chain gene on chromosome 7. The location of the variable (V), joining(J) and constant (C) regions are indicated. Colored arrows represent conserved regions within the variable region gene segments targetedby primers. Primers are represented by arrows with the size range of valid products generated with each of the master mixes indicatedbelow the figure. Colors correspond to the peak colors assigned to products when differential fluorescence detection methods are used.

Differential Fluorescence Detection
Differential fluorescence detection is commonly used to resolve the different-sized amplicon products using acapillary electrophoresis instrument. Primers can be conjugated with several different fluorescent dyes
(fluorophores) so that they can produce different emission spectra upon excitation by a laser in the capillaryelectrophoresis instrument. In this manner, different fluorescent dyes can correspond to different targeted regions.
This detection system results in unsurpassed 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, can virtually be eliminated. Further, differential detection allows accurate, reproducible
and objective interpretation of primer-specific products and automatic archiving of data. Inter-assay and intra-assay
reproducibility in size determination using capillary electrophoresis is approximately 1 to 2 base pair (bp). This
reproducibility and sensitivity coupled with the automatic archiving of specimen data allows for the monitoring,
tracking and comparison of data over time.

T-Cell Receptor Gamma Gene Rearrangement Assay

For RESEARCH USE ONLY; not intended for diagnostic use.

280280 Rev. G | September 2023

Page 5 of 17

4. Reagents
4.1.

Reagent Components

Table 1. Available Kits

Catalog #12070051

Description

Total Reactions

T-Cell Receptor Gamma Gene Rearrangement Assay - ABI Fluorescence Detection

30 Reactions

Table 2. Reagent Components

Reagent

Catalog #()
40880490

Control and Standard
40920010

Reagent Components(active ingredients)
IVS-0009 Clonal Control DNA

200 µg/mL of DNA in 1/10th TE solution

IVS-0000 Polyclonal Control DNA

200 µg/mL of DNA in 1/10th TE solution

Unit of
Quantity# of Units

100 µL

1

Storage Temp.

or
100 µL

1

1500 µL

11500 µL

11500 µL

1

Differential Fluorescence Detection
T-Cell Receptor Gamma Mix 1 – 6FAM

22070071
Master Mixes

T-Cell Receptor Gamma Mix 2 - HEX

22070021

Template
Amplification Control
Master Mix
Note :

Multiple oligonucleotides targeting the Vγ1-8 + Vγ9 +Jγ1/2 consensus regions of the T-cell receptor gamma
gene in a buffered salt solution.

20960011

Multiple oligonucleotides targeting the alternate Vγ +Jγ1/2 consensus regions of the T-cell receptor gamma
gene in a buffered salt solution.

Amplification Control Master Mix – 6FAM

Multiple oligonucleotides targeting the HLA-DQα locus.

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

T-Cell Receptor Gamma Gene Rearrangement Assay

For RESEARCH USE ONLY; not intended for diagnostic use.

280280 Rev. G | September 2023

Page 6 of 17

4.2.

Warnings and Precautions


















4.3.

Use this assay kit as a system; do not substitute other manufacturers’ reagents. Dilution, reducing amplification
reactions or other deviations from this protocol may affect the performance of this test and/or nullify any limited
sublicense that come with the purchase of these kits.
Do not mix or combine reagents from kits with different lot numbers.
Materials are stable until the labeled expiration date when stored and handle as directed. Do not use kitsbeyond their expiration date.
Adherence to the protocol will assure optimal performance and reproducibility. Ensure correct thermal cycler
programs are used, as other programs may provide inaccurate/faulty data such as false-positive and falsenegative results.
Dispose of unused reagents and waste in accordance with country, federal, state and local regulations.
Perform all laboratory procedures with standard personal protective equipment (gloves, laboratory coats andprotective eyewear). Follow good laboratory practices and universal precautions when working with specimens.
Do not pipette by mouth. Do not eat, drink or smoke in laboratory work areas. Wash hands thoroughly after
handling specimens and assay reagents. Handle specimens in approved biological safety containment facilities
and open only in certified biological safety cabinets.
Due to the high analytical sensitivity of this test, use extreme care to avoid any contamination of reagents oramplification mixtures with samples, controls or amplified materials. Use fresh, aerosol-resistant pipette tips
between samples and between dispensing reagents. Closely monitor all reagents for signs of contamination(e.g., negative controls giving positive signals). Discard any reagents suspected of contamination.
To minimize contamination, wear clean gloves when handling samples and reagents and routinely clean work
areas and pipettes prior to setting up PCR.
Follow uni-directional workflow between separate work areas in the PCR laboratory: begin with master mix
preparation, move to specimen preparation, then to amplification and finally to detection. Autoclaving does not
eliminate DNA contamination. Perform pre- and post-PCR steps in separate spaces. Avoid taking paper andother materials from post-PCR into the pre-PCR space.
Dedicate all pipettes, pipette tips and any equipment used in a particular area to that area of the laboratory.
Decontaminate non-disposable items with 10% bleach and rinse with distilled water two separate times before
returning them to the starting areas.
Use sterile, disposable plastic ware whenever possible to avoid contamination.

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

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

T-Cell Receptor Gamma Gene Rearrangement Assay

For RESEARCH USE ONLY; not intended for diagnostic use.

280280 Rev. G | September 2023

Page 7 of 17

5. Instruments
5.1.

Thermal cyclerUse or function: Amplification of DNA samples
Suggested instrument: Veriti™ thermal cycler or equivalent
Performance characteristics and specification:
o
o



5.2.

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

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

ABI Capillary Electrophoresis Instruments

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 each polymer and capillary type.
See section 6.4. Sample Preparation for sample preparation.

T-Cell Receptor Gamma Gene Rearrangement Assay

For RESEARCH USE ONLY; not intended for diagnostic use.

280280 Rev. G | September 2023

Page 8 of 17

6. 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.

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.

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

Sample Preparation

6.4.1.
6.4.2.

Test all samples in singlicate.
Include positive, negative and no template controls with each master mix tested.
Using any method of DNA extraction, extract the gDNA from unknown samples.
Resuspend DNA to final concentration of 100 µg – 400 µg per mL in TE (10 mM Tris-HCl, 1 mM EDTA, pH 8.0) or
distilled water.



6.5.

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, a template amplification control master mix (e.g., Amplification Control orSpecimen Control Size ladder) can be used to verify the absence of PCR inhibitors and sufficient quality andquantity of DNA is present to generate a valid result.

Template Amplification Control
The Amplification Control master mix primers are available unlabeled for gel detection and labeled with afluorescent dye (6-FAM) for ABI fluorescence detection. The 6-FAM label is detected as BLUE using the differential
fluorescence software. The amplicons produced with this master mix are 235 bp.


6.6.

Run the products of this master mix separately.

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

T-Cell Receptor Gamma Gene Rearrangement Assay

For RESEARCH USE ONLY; not intended for diagnostic use.

280280 Rev. G | September 2023

Page 9 of 17

7. Assay Procedure
7.1.

Materials Provided
Table 3. Materials Provided

Catalog #7.2.

Description

22070071

T-Cell Receptor Gamma Mix 1 – 6FAM

22070021

T-Cell Receptor Gamma Mix 2 – HEX

20960011

Amplification Control – 6FAM

40880490

IVS-0009 Clonal Control DNA

40920010

IVS-0000 Polyclonal Control DNA

Materials Required (not provided)

Table 4. Materials Required (not provided)

Reagent/Material
DNA Polymerase

Recommended Reagents/Materials and Suppliers
Roche:•
EagleTaq DNA Polymerase
Invivoscribe, Inc.
•
FalconTaq DNA Polymerase

Part Number
05206944190

Notes
N/A

60970130

Molecular Biology
Grade or USP Water

N/A

N/A

DNase / RNase free

Calibrated Pipettes

N/A

N/A

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

N/A

N/A

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 2600, PE 9600, or PE 9700

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

N/A

ABI Fluorescence Detection
ABI Capillary
Electrophoresis
Instrument

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

Hi-Di Formamide

Thermo Fisher Scientific:• Hi-DiTM Formamide

Size Standards

Invivoscribe, Inc.:o Hi-Di Formamide w/ROX size standards for ABI 310
o 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 GeneScan - 600 [LIZ]TM v2.0

T-Cell Receptor Gamma Gene Rearrangement Assay

4311320N/A

60980051
60980061
N/A
4029854408399

For RESEARCH USE ONLY; not intended for diagnostic use.

280280 Rev. G | September 2023

Page 10 of 17
Table 4. Materials Required (not provided)

Reagent/Material

Recommended Reagents/Materials and Suppliers

Spectral Calibration
Dye Sets

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)Polymer

BufferPart Number

4345827
402996

N/A4015464345833

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:o
10X Genetic Analyzer Buffer with EDTA

402824

T-Cell Receptor Gamma Gene Rearrangement Assay

Notes

40283843163554352755

For RESEARCH USE ONLY; not intended for diagnostic use.

N/A

Dilute 1:10 in sterile water
before use

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7.3.

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 clean, sterile
microfuge tube.


7.3.3.

Add the appropriate amount of Taq DNA polymerase (0.25 µL @5 U/ µL per 55 µL total reaction volume) toeach master mix and gently mix by inverting several times or gently vortexing.


7.4.

Aliquot volumes are 50 µL for each sample + 150 µL (3 x 50 µL) for the positive, negative and no templatecontrols.
Add an additional 20 µL to correct for pipetting errors.

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

Amplification
7.4.1.
7.4.2.
7.4.3.

Aliquot 50 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 appropriate PCR program in Table 5.


Use the calculated option for temperature measurement with the PTC instruments.
Table 5. Thermal cycling conditions

7.4.4.

7.5.

CyclesStep
1

Temperature
95ᐤC

Duration
7 minutes

2

94ᐤC

30 seconds

3

55ᐤC

30 seconds

4

72ᐤC

1 minute

572ᐤC

10 minutes

1

6

4ᐤC

∞

1

1
35

Remove the amplification plate from the thermal cycler.

Detection

ABI Fluorescence Detection with ABI 310, 3100 & 3130 instruments
7.5.1.
7.5.2.
7.5.3.

In a new microcentrifuge tube, mix an appropriate amount (10 μL per reaction) of Hi-Di Formamide with ROX
Size Standards.a Vortex well.
In a new 96-well PCR plate, add 10 μL of 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.


7.5.4.
7.5.5.
7.5.6.
7.5.7.
7.5.8.

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 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.
Review profile and controls, report results.
Data are automatically displayed as size and color specific peaks.

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.

T-Cell Receptor Gamma Gene Rearrangement Assay

For RESEARCH USE ONLY; not intended for diagnostic use.

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7.5.9.
7.5.10.
7.5.11.

In a new microcentrifuge tube, mix an appropriate amount (9.5 μL per reaction) of Hi-Di Formamide with LIZ
Size Standards.a Vortex well.
In a new 96-well PCR plate, add 9.5 μL of 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.


7.5.12.
7.5.13.
7.5.14.
7.5.15.
7.5.16.

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, report results.
Data are automatically displayed as size and color specific peaks.

Note:

Please see the accompanying Applied Biosystems product insert for mixing Hi-Di Formamide with size standards fordifferent ABI instruments.

7.6.

Recommended Positive ControlsaTable 6. Recommended positive controls and size of amplified products

Master Mix

TargetColorControl DNA

Catalog #Product Size (bp)

T-Cell Receptor
Gamma Mix 1Vγ1-8, 9 + J1/2
consensus

Blue

Valid Size Range – Group I
Valid Size Range – Group II
IVS-0009 Clonal Control DNA----40880490

55 – 85
155 – 185
601, 68

T-Cell Receptor
Gamma Mix 2

Alternate Vγ +
J1/2

Green

Valid Size Range – Group I
Valid Size Range – Group II
IVS-0009 Clonal Control DNA----40880490

200 – 2352
235 – 2702
2161, 248

Amplification
Control

HLA-DQαBlue

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

235
235

Note:
1Note:

2Note:

The amplicon sizes listed above were determined using an ABI platform. Amplicon sizes observed on each specific CE instrument may differ
1-4 bp from those listed above depending on the platform of detection (ABI) and the version of the analysis software used. Once identified,the amplicon size as determined on the specific platform used will be consistent from run to run.
These are generally observed as peaks with weak amplitudes.
This multiplex master mix generates two distinct size ranges of products (200-235 bp and 235-270 bp). A clonal sample will frequently generatea clonal product in each of these size ranges. For example, the positive control generates peaks of 216 and 248 bp. When products are
generated in both of these size ranges the difference in size of these peaks is approximately 32 bp.

T-Cell Receptor Gamma Gene Rearrangement Assay

For RESEARCH USE ONLY; not intended for diagnostic use.

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8. 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 NGS may be advisable to rule out clonality.
The size range for each master mix has been determined by testing positive control samples and gDNA. 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.

8.1.

Sample Analysis
Results can be reported as “Positive” or “Negative” for “Detection of clonal T-cell receptor gamma 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.
The following describes the analysis of each control and the decisions necessary based upon the results.







8.2.

Negative Control:(Polyclonal control, water or no template blank). If the negative control is:oPositive:

Possible contamination of all PCR amplification reactions. Do not continue with
the interpretation of results. Prepare fresh master mix and repeat
amplification.

oNegative:

Continue with the analysis.

Positive Control:(This can also be an extraction control if positive control material is taken
through extraction processes). If the positive control is:ooPositive:

Continue with analysis.

Negative:

Repeat the assay.

ooGenerates 235 bp product:

Continue with analysis.

Does not generate specified product:

Repeat the assay and/or re-extract the specimen.

Amplification Control:(This can be run on unknown samples). If the Amplification Control :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 T-cell receptor gamma chain gene rearrangement,
consistent with the presence of a clonal cell population.”

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

T-Cell Receptor Gamma Gene Rearrangement Assay

For RESEARCH USE ONLY; not intended for diagnostic use.

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10. Expected Results
10.1.

Expected Size of Amplified Products

Note:“Color” indicates the color of products generated with the master mix when using differential fluorescence
detection format (e.g., ABI instruments).

Table 7. Expected Size of Amplified Products

Master Mix

TargetT-Cell Receptor
Gamma Mix 1Vγ1-8,9 +T-Cell Receptor
Gamma Mix 2

Alt. Vγ+

Amplification
Control
Note:
ᐃNote:

ᕫNote:

10.2.

Jγ1/2

Jγ1/2
HLA-DQα

ColorControl DNA

Cat#

Product Size (bp)Blue

Valid Size Range – Group I
Valid Size Range – Group II
IVS-0000 Polyclonal Control DNA
IVS-0009 Clonal Control DNA----40920010
40880490

55 - 85
155 - 185
55 - 85, 155 - 185
60ᐃ, 68

Green

Valid Size Range – Group I
Valid Size Range – Group II
IVS-0000 Polyclonal Control DNA
IVS-0009 Clonal Control DNA----40920010
40880490

200 - 235ᕫ
235 - 270ᕫ
200 - 270
216ᐃ, 248

Blue

Valid Size
IVS-0000 Polyclonal Control DNA--40920010

235
235

The amplicon sizes listed above were determined using an ABI platform. Amplicon sizes observed on each specific CE instrument may differ 1-4
bp from those listed above depending on the platform of detection (ABI, etc.) and the version of the analysis software used. Once identified, theamplicon size as determined on the specific platform used will be consistent from run to run.
These are generally observed as peaks with weak amplitudes.
This multiplex master mix generates two distinct size ranges of products (200-235 bp and 235-270 bp). A clonal sample will frequently generatea clonal product in each of these size ranges. For example, the positive control generates peaks of 216 and 248 bp. When products are
generated in both of these size ranges the difference in size of these peaks is approximately 32 bp.

Sample Data

ABI Fluorescence Detection: The data shown in Figures 2 -4 were generated using the master mixes indicated. Amplified products were run on an ABI
instrument.

Figure 2.

T-Cell Receptor Gamma Gene Rearrangement Assay

For RESEARCH USE ONLY; not intended for diagnostic use.

Amplified products using the TCell Receptor Gamma Mix 1 –6FAM. (left)

280280 Rev. G | September 2023

Page 15 of 17

Figure 3.

Amplified products using the T-Cell Receptor
Gamma Mix 2 – HEX. (right)

Figure 4.

Data was generated by the
Amplification Control master mix.
(left)

11. Technical and Customer Service
Thank you for purchasing our TCRG Gene Rearrangement Assay. We appreciate your business. We are happy to assist you
in the validation 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

12. References
1.
2.
3.

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-CT983936. Leukemia. 2007; 21(2):201-6.

T-Cell Receptor Gamma Gene Rearrangement Assay

For RESEARCH USE ONLY; not intended for diagnostic use.

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Page 16 of 17

13. Symbols
The following symbols are used in Invivoscribe product labeling.
Catalog Number

Expiration Date

Reagent Volume

Manufacturer

Lot Number

Consult Instructions for Use

Storage Conditions

Research Use Only

14. 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 the product 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 no liability 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 and continually monitored through purchaser defined andcontrolled processes including but not limited to testing of positive, negative, and blank controls every time a sample is tested.
Ordering, acceptance, and use of product constitutes purchaser acceptance of sole responsibility for assuring product efficacy
and purchaser agreement to the limitation 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, and related pending andfuture 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 of these products require nucleic acid amplification
methods such as Polymerase Chain Reaction (PCR). No license under these 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.

T-Cell Receptor Gamma Gene Rearrangement Assay

For RESEARCH USE ONLY; not intended for diagnostic use.

280280 Rev. G | September 2023

Page 17 of 17

15. T-Cell Receptor Gamma Gene Rearrangement Assay: Single Page Guide
15.1.

Using gloved hands, remove the master mixes from the freezer. Allow the tubes to thaw; then gently vortex to mix.

15.2.

In a containment hood or dead air box remove an appropriate aliquot to clean, sterile microfuge tube (one tube for eachmaster mix). Aliquot volumes are 50 µL for each sample + 150 µL for the positive, negative and no template controls. Add an
additional 20 L to correct for pipetting errors.

15.3.

Add the appropriate amount of Taq DNA polymerase (0.25 µL @5 U/ µL per 55 µL total reaction volume) to each master mix
and gently mix by inverting several times or gently vortexing.

15.4.

Aliquot 50 µL of master mix to individual wells of a PCR plate.

15.5.

Add 5 µL of 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.

ABI Fluorescence Detection with ABI 310, 3100 & 3130 instruments

15.6.

In a new microcentrifuge tube, mix an appropriate amount (10 μL per reaction) of Hi-Di Formamide with ROX Size Standards.
Vortex well.

15.7.

In a new 96-well PCR plate, add 10 μL of Hi-Di Formamide with ROX size standards to individual wells for each reaction.

15.8.

Transfer 1 μL of each reaction to the wells containing Hi-Di Formamide with ROX size standards. Add only one sample perwell. Pipette up and down to mix.

15.9.

Cap or cover the PCR plate.

15.10. Heat denature the samples at 95ºC for 2 minutes, then snap chill on ice for 5 minutes.
15.11. Prepare a sample sheet and injection list for the samples.
15.12. Run the samples on an ABI 310/3100/3130 capillary electrophoresis instrument according to its user manual.
15.13. Review profile and controls, then report results.
ABI Fluorescence Detection with ABI 3500 instruments

15.14. In a new microcentrifuge tube, mix an appropriate amount (9.5 μL per reaction) of Hi-Di Formamide with LIZ Size Standards.
Vortex well.

15.15. In a new 96-well PCR plate, add 9.5 μL of Hi-Di Formamide with LIZ size standards to individual wells for each reaction.
15.16. Transfer 0.5 μL of each reaction to the wells containing Hi-Di Formamide with LIZ size standards. Add only one sample perwell. Pipette up and down to mix.

15.17. Cap or cover the PCR plate.
15.18. Heat denature the samples at 95ºC for 3 minutes, then snap chill on ice for 5 minutes.
15.19. Prepare a sample sheet and injection list for the samples.
15.20. Run the samples on an ABI 3500 capillary electrophoresis instrument according to its user manual.
15.21. Review profile and controls, then report results.

T-Cell Receptor Gamma Gene Rearrangement Assay

For RESEARCH USE ONLY; not intended for diagnostic use.

280280 Rev. G | September 2023