REF RQ-115-4M (50 tests)

REF RQ-115-6M (100 tests)

REALQUALITY
RQ-BCR-ABL p190
One-Step

Kit for detection and quantification of translocation

t(9;22)(q34;q11),
in variant p190,
by one-step Real-Time RT-PCR

USER MANUAL

AB ANALITICA srl
Via Svizzera 16 - 35127 PADOVA, ITALY
Tel +39 049 761698 - Fax +39 049 8709510
e-mail: info@abanalitica.it

RQ-BCR-ABL p190_One-Step_manual_e20161209
1 PRODUCT INFORMATION 2
1.1 Intended use 2
2 KIT CONTENT 3
3 STORAGE AND STABILITY OF REAGENTS 3
4 PRECAUTIONS FOR USE 4
5 SAFETY RULES 5
5.1 General safety rules 5
5.2 Safety rules concerning the kit 5
6 REQUIRED, BUT NOT INCLUDED MATERIALS 5
6.1 Reagents 5
6.2 Instruments 5
6.3 Disposables 6
7 INTRODUCTION 7
8 TEST PRINCIPLE 8
9 PRODUCT DESCRIPTION 9
10 SAMPLE COLLECTION, MANIPULATION AND PRETREATMENT 9
11 PROTOCOL 10
11.1 RNA Extraction 10
11.2 Instrument programming 10
11.2.1 Thermal profile and fluorescence reading 10
11.2.2 Setup of samples and controls/standards 10
11.3 Preparation of the reaction mix 11
11.4 Analysis of results 12
11.4.1 Verifying the run 13
11.4.2 Interpretation of results 14
12 TROUBLESHOOTING 15
13 DEVICE LIMITATIONS 16
14 DEVICE PERFORMANCE 16
14.1 Analytical specificity 16
14.2 Analytical sensitivity: Limit of detection (LoD) 16
14.3 Analytical sensitivity: linear range 16
14.4 Reproducibility 16
14.5 Diagnostic specificity 16
14.6 Diagnostic sensitivity 17
14.7 Accuracy 17
15 REFERENCES 18
15.1 Websites 18
16 RELATED PRODUCTS 19
17 SYMBOLS 20

RQ-BCR-ABL p190_One-Step_manual_e20161209 1
1 PRODUCT INFORMATION

1.1 Intended use

The REALQUALITY RQ-BCR-ABL p190 One-Step kit is an IVD for detection and quantification of variant
p190 (m-bcr transcript e1a2) of translocation t(9;22)(q34;q11), which involves the ABL proto-oncogene on
chromosome 9 and a part of the BCR gene on chromosome 22. The test is based on PCR amplification on
cDNA of the BCR-ABL fusion gene.
If used in combination with the REALQUALITY RQ-BCR-ABL p190 STANDARD, code RQ-116-SM, it allows
absolute quantification of the M-bcr transcripts present in the sample.
The test is based on reverse transcription and Real-Time PCR in a single step on RNA extracted from
human clinical samples.
This in vitro diagnostic test allows the detection and quantification of m-bcr and is an auxiliary device for
diagnosis and monitoring of clinical syndromes associated with the Philadelphia chromosome (Ph+), such as
Chronic Myeloid Leukemia (CML) and Acute Lymphoblastic Leukemia (ALL). It is recommended to use this
kit as indicated in the instructions herein.

This manual refers to the following product:

REALQUALITY RQ-BCR-ABL p190 One-Step

Kit for detection and quantification of translocation t(9;22)(q34;q11), in variant p190, by one-step Real-Time
RT-PCR.
This product is in accordance with directive 98/79/EC (Annex III) regarding in vitro medical diagnostic
devices (CE marking).
Contains all reagents needed for reverse transcription and Real-Time PCR.

Code Product PKG

RQ-115-4M REALQUALITY RQ-BCR-ABL p190 One-Step 50 tests

RQ-115-6M REALQUALITY RQ-BCR-ABL p190 One-Step 100 tests

RQ-BCR-ABL p190_One-Step_manual_e20161209 2
 2 KIT CONTENT
BOX RG
STORE AT -30 °C TO -20 °C

TUBE (T)
DESCRIPTION LABEL or CAP 25 tests 50 tests 100 tests
COLOR

Master mix containing

BCR-ABL p190
reagents for reverse
One-Step Green 1 × 472.5 µL 2 × 472.5 µL 4 × 472.5 µL
transcription and amplification
Real time mix
of BCR-ABL p190
Master mix containing
ABL
reagents for reverse
One-Step Blue 1 × 472.5 µL 2 × 472.5 µL 4 × 472.5 µL
transcription and amplification
Real time mix
of ABL

Mix containing DNA

polymerase and reverse RT Enzyme mix - 1 × 135 µL 2 × 135 µL 4 × 135 µL
transcriptase

BOX PC
STORE AT -30 °C TO -20 °C

TUBE (T)
DESCRIPTION LABEL or CAP 25 tests 50 tests 100 tests
COLOR

Positive control for m-bcr

transcript e1a2 and for ABL PC
(DNA containing parts of the BCR-ABL Green 1 × 50 µL 2 × 50 µL 4 × 50 µL
BCR-ABL p190 and ABL p190/ABL
sequences)

3 STORAGE AND STABILITY OF REAGENTS

Each component of the kit must be stored according to the indications on the label of the corresponding box.

BOX RG Store at -30 °C to -20 °C

BOX PC Store at -30 °C to -20 °C

If stored at the recommended temperature, all reagents are stable until the expiration date reported on the
packaging.

The Real time mixes are sensitive to change of physical state. They should NOT undergo more than two
freeze/thaw cycles. If performing runs with low numbers of samples, it is recommended to aliquot the
reagents beforehand.
The Real time mixes contain fluorescent molecules and should be stored protected from light.

The positive control is sensitive to change of physical state. It should NOT undergo more than three
freeze/thaw cycles. If performing runs with low numbers of samples, it is recommended to aliquot the
reagents beforehand.

RQ-BCR-ABL p190_One-Step_manual_e20161209 3
4 PRECAUTIONS FOR USE
 The kit must be used only as an IVD and be handled by qualified technicians who are trained in
techniques of molecular biology applied to diagnostics;

 Before starting the kit procedure read the user manual carefully and completely;

 Keep the kit protected from heat and direct light;

 Please pay particular attention to the expiration date on the label of each box. Do not use any part of the
kit past the expiration date;

 The reagents present in the kit must be considered an undividable unit. Do not use them separately or in
combination with reagents from other kits or lots;

 The Real time mixes must be thawed at room temperature before use. After thawing, mix the solutions
by inverting the tubes several times. Do not vortex! Then centrifuge briefly;

 To avoid degradation of the RT Enzyme mix, store it at -30 °C to -20 °C until use and return it to -30 °C
to -20 °C immediately after use. Before use, mix the solution by inverting the tubes several times. Do not
vortex! Then centrifuge briefly;

 The positive control (PC) must be thawed at room temperature before use. Mix the solution well, then
centrifuge briefly;

 Work quickly, particularly if preparing the reactions at room temperature. If possible work on ice or on a
cooling block.

In case of any doubt about storage conditions, box integrity or application of the method, please contact the
technical support team at AB ANALITICA:
laboratorio@abanalitica.it

For nucleic acid amplification the user has to take the following precautions:

 Make sure that the analysis procedure is performed in absence of RNases and DNases;

 Use filter-tips;

 In order to avoid contamination store biological samples, extracted RNA, PCR product and the positive
control included in the kit separate from the Real time mixes and the RT Enzyme mix;

 Set up pre- and post-PCR areas. Do not share instruments or consumables (pipettes, tips, tubes, etc.)
between those areas;

 Change gloves frequently;

 Wash the bench surfaces with 5 % sodium hypochlorite;

 Keep RNA on ice during preparation of the RT-PCR;

Note: If the reverse transcription is not performed immediately, store the RNA at -80 °C. For a short
period, RNA can also be stored at -30 °C to -20 °C.

RQ-BCR-ABL p190_One-Step_manual_e20161209 4
5 SAFETY RULES

5.1 General safety rules

 Wear disposable gloves when handling reagents and clinical samples. Wash hands after the procedure;

 Do not pipet by mouth;

 No known diagnostic method can assure the absence of infective agents. Therefore, consider all clinical
samples to be potentially infectious and handle them accordingly;

 All devices that come into contact with clinical samples must be considered contaminated and disposed
of as such. In case of accidental spilling of samples clean up with 10 % sodium hypochlorite. Materials
you use to clean must be disposed of in special containers for contaminated products;

 Clinical samples, contaminated materials and products must be decontaminated before disposal.
It is recommended to use one of the following decontamination methods:
a) immerse for 30 minutes in a solution of 5% sodium hypochlorite
(1 volume of 5 % sodium hypochlorite solution on 10 volumes of contaminated fluid);
b) autoclave at 121 °C for at least 2 hours (ATTENTION! Do not autoclave solutions containing sodium
hypochlorite!!).

5.2 Safety rules concerning the kit

The risks for use of this kit are related to the single components.

Dangerous components: none.

The Material Safety Data Sheet (MSDS) of this device is available upon request.

6 REQUIRED, BUT NOT INCLUDED MATERIALS

6.1 Reagents

 Erythrocyte lysis buffer containing ammonium chloride;

 Reagents for RNA extraction;

 DNAse- and RNAse-free sterile water;

 For quantitative analysis: REALQUALITY RQ-BCR-ABL p190 STANDARD, code RQ-116-SM.

6.2 Instruments

 Laminar flow cabinets:

use while preparing the amplification mix in order to avoid contamination. It is recommended to use a
different laminar flow cabinet when adding RNA extract and positive control / quantification standards;
 Micropipettes (range: 0.5 - 10 µL, 2 - 20 µL or 10 - 100 µL);

 Microcentrifuge (max. 12,000-14,000 rpm);

 Plate centrifuge (optional);

 Real-Time PCR instrument

RQ-BCR-ABL p190_One-Step_manual_e20161209 5
 This kit has been validated on:
 Applied Biosystems 7500 Fast/7500 Fast Dx Real-Time PCR System (ABI 7500 Fast/Fast Dx –
Applied Biosystems)
The kit can be used on instruments that work with 25 μL of reaction volume and can read the
fluorescence of the FAM. Compatibility of the device with other commercially available instruments has
been asserted. For further information on instrument compatibility please contact the technical support
team at AB ANALITICA.

6.3 Disposables

 Talc-free disposable gloves;

 Disposable sterile filter-tips (range: 0.5 - 10 µL, 2 - 20 µL or 10 - 100 µL);

 96-well plates for Real-Time PCR and adhesive optical film.

RQ-BCR-ABL p190_One-Step_manual_e20161209 6
7 INTRODUCTION
Studies of leukemia and lymphomas have opened the way to a better understanding of the cellular and
molecular mechanisms behind many other neoplastic diseases. In contrast to solid tumors that require
invasive sampling techniques, leukemic cells can be easily extracted from blood samples. So, leukemia and
lymphomas had become some of the best-studied neoplastic diseases.
The chromosomal rearrangement known as the Philadelphia chromosome (Ph) was the first clonal marker to
be identified in a neoplastic disease (Nowell and Hunderford, 1960) and the molecular events underlying the
Philadelphia chromosome translocation were among the first oncogene translocations to be reported in
human tumors (Groffen et al., 1984).

The Ph chromosome derives from translocation t(9;22)(q34;q11) and is present in more than 95 % of cases
of Chronic Myeloid Leukemia (CML). It is also found in 5 % of Acute Lymphoblastic Leukemia (ALL) in
children and 10 - 25 % of ALL cases in adults. For both, ALL and CML presence of this translocation is a
marker for a negative prognosis.
On the molecular level, the translocation juxtaposes the proto-oncogene ABL (c-ABL) from chromosome 9 to
a specific region in the BCR gene on chromosome 22. The chromosomal breakpoint in the ABL gene is
located in exon 2. In contrast, the breakpoints of the BCR gene can be located in different regions, giving
rise to different fusion transcripts.

In majority of the CML cases, in 30 % to 50 % of Ph+ ALL cases in adults and in 20 % to 30 % of Ph+ ALL
cases in children, the breakpoint of the BCR gene occurs in the so-called “major breakpoint cluster region M-
bcr”. This region is located between exon 12 and 16 (also known as exon b1-b5). Most frequently
breakpoints are found in exons b2 and b3 of the BCR gene and exons a2 and a3 of the ABL gene. Virtually
all CML-affected patients carry the b3a2 (55 %) or b2a2 (40 %) translocation. The less frequent b3a3 and
b2b3 translocations derive from alternative splicing (van Dongen et al., 1999) and have been found only in a
small portion of patients (5 %).
The BCR-ABL M-bcr fusion gene gives rise to a fusion protein of 210 kDa (BCR-ABL p210), which displays
transforming activity, stimulating uncontrolled cell proliferation (Melo, 1996; Verfaillie, 1998).

Another breakpoint in the BCR gene is almost exclusively found in Ph+ ALL patients. Roughly 60 % of all
Ph+ ALL cases are characterized by this breakpoint, which is located in the “minor breakpoint cluster region
m-bcr” of the BCR gene. In these cases, the translocation juxtaposes BCR exon e1 and ABL exon a2 (e1-a2
rearrangement). The resulting fusion gene BCR-ABL m-bcr produces a 190 kDa fusion protein (BCR-ABL
p190).
Although the e1-a2 transcript is mainly associated with ALL, about ten cases of CML have been reported
that were characterized by the exclusive presence of the BCR-ABL m-bcr transcript and the BCR-ABL p190
protein. Moreover, it has been demonstrated that, owed to alternative splicing, virtually all CML cases that
are positive for the BCR-ABL p210 transcript, also show low levels of e1-a2 transcript. The clinical and
pathogenetic significance of this phenomenon is still not clear.

Occasionally, a fusion transcript named BCR-ABL µ-bcr is found in CML patients. This transcript derives
from the juxtaposition of BCR exon 19 and ABL exon 2. It is translated into a 230 kDa fusion protein
(BCR-ABL p230) which is appears to impair the differentiation process of granulocytes, resulting in a
particularly severe course of the disease.
Some sporadic CML cases have been described, that exhibit BCR breakpoints located outside of the three
previously mentioned regions, involving other introns (2, 5, 6, 8, and 10), instead. Additionally, in rare
Ph+ CML and ALL cases, the ABL breakpoint has been found in exon a3 instead of exon a2.

The translocation can be detected at the molecular level by extracting total RNA from clinical samples,
reverse transcribing the RNA into cDNA and amplifying the gene regions of interest from the cDNA.
Detecting and identifying the translocation not only provides useful information for diagnosis and prognosis
of those types of leukemia, but also provides the means for monitoring the Minimal Residual Disease (MRD).
MRD describes the remainder of neoplastic cells that persists in the patient during the different phases of
chemotherapy and which cannot be detected by methods of cytology (e.g. analysis of cell morphology).
Even with aggressive chemotherapy a significant percentage of patients suffer a disease relapse at various
stages after starting the treatment. The relapse is often the result of the MRD, the persistence of therapy-
resistant cells. Therefore, a precise monitoring of MRD would be very valuable for the therapy of leukemia.

RQ-BCR-ABL p190_One-Step_manual_e20161209 7
Only since analysis techniques with higher sensitivity became available, these persistent cells can be
detected and characterized. The use of PCR has opened up new possibilities for extended and efficient
MRD monitoring (van Dongen et al., 1998; Baccarani et al., 2006).

8 TEST PRINCIPLE
The PCR method (Polymerase Chain Reaction) was the first method of DNA amplification described in
literature (Saiki RK et al., 1985). It is a method for in vitro amplification of a specific part of DNA (target
sequence) by use of a thermostable DNA polymerase.
This technique was shown to be a valuable and versatile instrument of molecular biology: its application
contributed to a more efficient study of new genes and their expression and has revolutionized for instance
the fields of laboratory diagnostics and forensic medicine.
The Real-Time PCR represents an advancement of this basic research technology, providing the possibility
to determine the number of amplified DNA molecules (amplicons) during the polymerase chain reaction
(PCR).

In the system at hand, monitoring the amplification progress is based on primers/probes labeled with
fluorescent molecules. These probes contain a reporter fluorophore and a molecule (quencher) that blocks
the reporter’s specific fluorescence. The fluorescent emission of the reporter is determined by its distance to
the quencher. As long as a probe is not bound to a target sequence, the reporter and quencher are in close
proximity and the reporter’s fluorescence is blocked. Upon binding to a target sequence, quencher and
reporter become separated and the reporter can emit fluorescent light, which is detected.

Typically, the main part of a Real-Time PCR run consists of 30 to 50 amplification cycles. A thermocycler
equipped with an adequate detector can record the fluorescence events at each cycle and thus monitor the
reaction in “real time”.
The cycle at which the fluorescence of the amplification product becomes clearly distinguishable from the
background is specific for each reaction and is correlated to the initial concentration of the target sequence.
This cycle is called threshold cycle (Ct). This Ct value is used to determine the initial target concentration,
with the help of a standard curve. A standard curve is created by amplification of solutions with known
concentrations of the target sequence (Fig. 1).

Fig. 1: Creating a standard curve using standards with known

concentrations.

RQ-BCR-ABL p190_One-Step_manual_e20161209 8
The main advantage of the Real Time PCR compared to conventional techniques of amplification is the
possibility to perform a semi-automated amplification. This means, the extra steps necessary to visualize the
amplification result are be avoided and the risk of contamination by post-PCR manipulation is reduced.

9 PRODUCT DESCRIPTION
The REALQUALITY RQ-BCR-ABL p190 One-Step kit, code RQ-115, is an IVD for detection and
quantification of variant p190 (m-bcr transcript e1a2) of translocation t(9;22)(q34;q11).
If used in combination with REALQUALITY RQ-BCR-ABL p190 STANDARD, code RQ-116-SM, calibrated
with the certified plasmid reference material IRMM ERM-AD623 BCR-ABL1, it allows quantification of m-bcr
transcripts in the sample and normalization to the expression of the housekeeping gene ABL.
The gene products are quantified using five-point standard curves (100 to 1,000,000 copies/reaction) for the
amplification targets BCR-ABL p190 and ABL.
Using the same RNA extract in a separate reaction (reverse transcription and PCR), transcripts of the
housekeeping gene ABL are amplified. Quantification of the ABL transcript not only allows normalization of
the BCR-ABL fusion transcript, but also permits the user to detect reaction inhibitors and to assess the
quality of the RNA extract. This is a valuable tool for identifying false-negative results.
Studies have shown that the amplification of targets with highly differing concentrations in the same reaction
may lead to a strong competition, which severely impedes amplification of the lower concentrated target and
results in false-negatives. For this reason, the amplification of ABL and BCR-ABL p190 is performed in
separate reactions.

The positive control supplied in this kit contains sequences of nucleic acid that corresponds to the amplified
transcript regions of BCR-ABL p190 and ABL, and is not harmful to the user.

The two ready-to-use master mixes included in the kit contain all reagents needed for the PCR as well as
ROX™, an inert colorant that exhibits stable fluorescent properties throughout all amplification cycles. On
some Real-time PCR instruments (Applied Biosystems, Stratagene etc.) it is used for normalization,
compensating differences between wells caused by pipetting errors or instrument limitations.
This kit was developed in accordance with the guidelines of Europe Against Cancer (Gabert et al., Leukemia
2003).

10 SAMPLE COLLECTION, MANIPULATION AND PRETREATMENT

Commonly whole peripheral blood or bone marrow is used for the analysis of BCR-ABL p190.
Sample collection should follow the common routine and respect all standard sterility precautions. Make sure
the blood is treated with EDTA. Do not use other anticoagulation agents, like heparin. They are strong
inhibitors of the Taq polymerase and may impair the Real-Time PCR.
Store fresh blood at +2 °C to +8 °C and extract the nucleic acids within 4 hours. Lyse erythrocytes using
ammonium chloride buffer. Proceed with the RNA extraction directly from the pellet containing the
leukocytes. If the extraction is not performed immediately, store the pellet at -80 °C. Before freezing, it is
recommended to resuspend the pellet in a buffer containing RNAse inhibitors (e.g. Trizol or RLT buffer by
QIAGEN).
The kit was validated on RNA extracted from leukocyte pellets.

RQ-BCR-ABL p190_One-Step_manual_e20161209 9
11 PROTOCOL

11.1 RNA Extraction

The device was validated for use with the most common manual or automated RNA extraction methods,
such as:
 RNeasy Mini Kit (QIAGEN)
For any further information on device compatibility with different extraction methods and validation, see the
attached Appendix 1; please contact the technical support team at AB ANALITICA.
The optimal RNA amount for PCR is 200 ng/reaction (40 ng/µL). If the RNA extract has a too high
concentration, dilute the extract with H2O DEPC water to 40 ng/µL.
The RNA concentration in the extract must be assessed by spectrophotometric absorption measurement at
260 nm. The A260/A280 ratio must lie between 1.8 and 2.1. Lower ratios indicate the presence of protein
contaminations in the RNA extract.

11.2 Instrument programming

11.2.1 Thermal profile and fluorescence reading

Set up the following thermal profile in your instrument:

Step Repeats Time (°C)
Reverse transcription 1 1 30:00 48.0
Taq Activation 2 1 10:00 95.0
00:15 95.0
Amplification cycles 3 50
01:00 60.0 *
* Fluorescence detection step

The fluorophore to be read for both BCR-ABL p190 and ABL is FAM.
Depending on the Real-Time PCR instrument select the appropriate channels:
Name Reporter Dye Quencher Dye

BCR-ABL p190 FAM None

ABI 7500 Fast/Fast Dx *

ABL FAM None

* For instruments that require a passive reference (e.g. Applied Biosystems, Stratagene) make sure to select
ROX™ for all wells in use.

Set the final reaction volume.

Attention: A more detailed protocol regarding the programming of different Real-Time PCR instruments can
be found in Appendix 2. Please contact the technical support team at AB ANALITICA and state the PCR
machine of interest.

11.2.2 Setup of samples and controls/standards

Set up samples, standards and controls in the instrument software at the corresponding positions. Name
each sample, control and standard using its proper name.
The quantitative analysis can be performed using the product REALQUALITY RQ-BCR-ABL p190
STANDARD, code RQ-116-SM. Enter the concentrations of the BCR-ABL p190 and ABL standards (100 to
1,000,000 copies/reaction [c/rx]).

RQ-BCR-ABL p190_One-Step_manual_e20161209 10
11.3 Preparation of the reaction mix

Thaw the reagents. Once thawed, homogenize the Real time mixes by inverting the tubes several times (do
NOT vortex!). Then centrifuge briefly.
Avoid degradation of the RT Enzyme mix. It must be stored at -30 °C to -20 °C until use and returned
to -30 °C to -20 °C directly after use. Before adding the RT Enzyme mix to the reaction mix: homogenize the
solution by inverting the tubes several times. Do not vortex! Then centrifuge briefly.
Work fast if preparing the reaction at room temperature. If possible, work on ice or a cooling block and in an
area protected from direct light.
Prepare one reaction master mix for BCR-ABL p190 and one reaction master mix for ABL, as specified
below (Tab. 1), that are sufficient for the analysis you want to perform, as indicated in Tab. 2, including at
least one excess sample volume as dead volume.

Tab. 1: Preparation of reaction master mixes for BCR-ABL p190 and ABL.

BCR-ABL p190 amplification * 1 Rx

BCR-ABL p190 One-Step Real time mix 17.5 μL
RT Enzyme mix 2.5 μL
PC BCR-ABL p190/ABL or quantification standard or RNA extract or negative control 5.0 μL

Total Volume 25.0 μL

ABL amplification * 1 Rx
ABL One-Step Real time mix 17.5 μL
RT Enzyme mix 2.5 μL
PC BCR-ABL p190/ABL or quantification standard or RNA extract or negative control 5.0 μL

Total Volume 25.0 μL

* ATTENTION! The PCRs for BCR-ABL p190 and ABL must be performed in separate wells.

RQ-BCR-ABL p190_One-Step_manual_e20161209 11
Tab. 2: Number of reactions to perform for qualitative or quantitative analysis of targets BCR-ABL p190 and
ABL.

QUALITATIVE ANALYSIS QUANTITATIVE ANALYSIS

target target target target

BCR-ABL p190 ABL BCR-ABL p190 ABL

N samples of RNA N samples of RNA

(duplicates) (duplicates)

PC BCR-ABL p190/ABL
––
(duplicates)

REALQUALITY
RQ-BCR-ABL p190 STANDARD
––
STANDARD 1, 2, 3, 4 and 5
(duplicates)

Negative control Negative control

(single or duplicates) (single or duplicates)

Homogenize the reaction master mixes by inverting the tubes several times (do NOT vortex!). Then
centrifuge briefly.
Pipet 20 µl of the reaction master mixes into the corresponding wells/tubes. Make sure to prepare sufficient
wells/tubes for all samples, the quantification standards or positive control and a negative control.
To each position add 5 µL of RNA extract (at 40 ng/µL) or, to the dedicated positions, add 5 µL of the
corresponding quantification standard / positive control (after thawing, homogenization and brief
centrifugation), or 5 µL of DNase- and RNase-free sterile water (negative control).
Make sure no air bubbles remain at the bottom of the wells and centrifuge at 4000 rpm for 1 minute.
Load the samples (plate, tubes etc.) on the instrument making sure to position/load them correctly and start
the RT-PCR run.

11.4 Analysis of results

After the PCR run is finished, view the analysis graph in logarithmic scale. Analyze the amplification results
separately for BCR-ABL p190 and ABL selecting the appropriate target and setting the threshold and
baseline values as follows:

INSTRUMENT TARGET CHANNEL BASELINE THRESHOLD

BCR-ABL p190 FAM 3-15 0.1
ABI 7500 Fast / Fast Dx
ABL FAM 3-15 0.1

Proceed with the interpretation of results as described in the following paragraphs.

RQ-BCR-ABL p190_One-Step_manual_e20161209 12
11.4.1 Verifying the run

Before interpreting the results of the clinical samples you need to verify the PCR run.

A. QUALITATIVE ANALYSIS: Verification of reaction controls

Perform the verification for both BCR-ABL p190 and ABL.

RESULT INTERPRETATION

Amplification signal Control and reaction suitable

PC BCR-ABL p190/ABL
Some amplification problems exist
No amplification signal
repeat the analysis

A contamination was introduced

Amplification signal
repeat the analysis
Negative control

No amplification signal Control and reaction suitable

The analysis session is valid if both controls for both reactions give "suitable" as result.

B. QUANTITATIVE ANALYSIS: Verification of reaction controls and standard curve

Perform the verification for both BCR-ABL p190 and ABL.
RESULT INTERPRETATION
A contamination was introduced
Amplification signal
repeat the analysis
Negative control

No amplification signal Control and reaction suitable

INSTRUMENT STANDARD CURVE PARAMETERS

-3.60 < slope < -3.10

ABI 7500 Fast / Fast Dx 2
R > 0.99

Note: If you work in duplicates: The Ct values of duplicates should not produce a difference of
Ct (ΔCt) >1.5.
The analysis session is valid if both the negative control and the parameters of the standard curve are
suitable.

RQ-BCR-ABL p190_One-Step_manual_e20161209 13
11.4.2 Interpretation of results

If the results of the controls are as expected, evaluate the samples according to the table below.
TARGET RESULT INTERPRETATION

Amplification signal Suitable

ABL
Not suitable
No amplification signal
Repeat RNA extraction

Positive
Amplification signal
for m-bcr transcript
BCR-ABL p190
Negative
No amplification signal
for m-bcr transcript

If amplifying in replicates: one BCR-ABL p190 positive replicate is sufficient to identify a clinical sample as
BCR-ABL p190 positive. Accordingly, a sample is only BCR-ABL p190 negative if all replicates are negative.

If at least one replicate is positive for BCR-ABL p190, you may use the standard curves for BCR-ABL p190
and ABL to calculate the copy numbers of BCR-ABL p190 (BCR-ABL p190 CN) and ABL (ABL CN).

For assessment of the deep Molecular Response (deep MR), it is highly recommended that each replicate of
the samples contains at least 10000 copies of ABL (Cross et al., 2015).

A precise quantification in regard to BCR-ABL p190 CN and ABL CN is only possible if the copy numbers lie in
the linear range of this device (see chapter 14 DEVICE PERFORMANCE).

Taking into consideration the theoric limit of detection (LoD) calculated by Real-Time PCR, it is
recommended to assign 3 copy numbers of the BCR-ABL p190 target for low positive samples (CN ≤ 3
copies/reaction), as indicated by the EUTOS document (EUTOS - European Treatment and Outcome Study
for CML-Version 4 August 2014).

The use of REALQUALITY RQ-BCR-ABL p190 STANDARD standard for quantification allows for the direct
quantification of cDNA samples without the need to elaborate the data further (White H et al. 2014; EUTOS -
European Treatment and Outcome Study for CML-Version 4 August 2014).

For quantification of the fusion transcript in the clinical sample the normalized copy number (NCN) of the
BCR-ABL p190 transcript needs to be calculated. Proceed as follows:

BCR-ABL p190 CN
NCN BCR-ABL % = × 100
ABL CN

If you have performed the PCR in replicates, calculate the sum of the copy numbers (CN) for the replicates
of BCR-ABL p190 and of ABL (Cross et al. 2014; EUTOS - European Treatment and Outcome Study for
CML - Version 4 August 2014; Cross et al., 2015):

(CN replicate 1) BCR-ABL p190 + (CN replicate 2) BCR-ABL p190

NCN BCR-ABL % = × 100
(CN replicate 1) ABL + (CN replicate 2) ABL

RQ-BCR-ABL p190_One-Step_manual_e20161209 14
12 TROUBLESHOOTING
No amplification signals for positive control / standards and samples

 The instrument was not programmed correctly.

 Repeat the amplification taking care of the instrument programming. Pay particular attention to the
thermal profile, the selected fluorophores and that the positions of the samples in the instrument setup
correspond to the actual sample positions/order.

 The reaction mix was not correctly prepared.

 Prepare a new reaction mix making sure to follow the instructions given in paragraph 11.3.

 The product was not stored correctly or used past the expiration date.
 Make sure the Real time mixes are stored at -30 °C to -20 °C and protected from light. Avoid
unnecessary freeze/thaw cycles.
 Make sure the RT Enzyme mix is stored at -30 °C to -20 °C until use and returned to -30 °C to -20 °C
immediately after use, in order to avoid degradation.
 Do not use the product past the expiration date.

Very late amplification signal for positive control / standards

 The positive control / quantification standards were not stored appropriately and have degraded.
 Always store the positive control / quantification standards at -30 °C to -20 °C and make sure they do not
undergo more than three freeze/thaw cycles.
 Do not use the positive control / quantification standards past the expiration date.

Amplification signal of ABL is very delayed or absent in the extracted sample (BCR-ABL p190
negative)

 The extracted RNA is not suitable for reverse transcription and/or amplification and the reaction was
inhibited.
 Make sure that the RNA extraction is performed correctly.
 If an extraction method uses washing steps with solutions containing ethanol, make sure no ethanol
residue remains in the sample.
 Use the extraction method(s) recommended in section 11.1.
 Make sure the RNA extract is correctly stored until use.
 Handle RNA according to the standard procedures for minimizing RNA degradation, like using RNase-
free plastic lab wear and working on ice while preparing the reverse transcription.

 The clinical sample is not suited for this analysis.

 Make sure the clinical sample was correctly pretreated before molecular analysis.

In case of any further problems, please contact the technical support team at
AB ANALITICA:
laboratorio@abanalitica.it
fax (+39) 049-8709510
tel. (+39) 049-761698

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13 DEVICE LIMITATIONS
The kit can have reduced performance if:

 The clinical sample is not suited for this analysis (blood treated with anticoagulation agents other than
EDTA, like heparin);

 The clinical sample was not treated as required (see chapter 10);

 The RNA is not suited for reverse transcription and/or amplification (e.g. presence of PCR inhibitors or
use of an incompatible extraction system);

 The kit was not stored correctly.

14 DEVICE PERFORMANCE

14.1 Analytical specificity

Specificity of the REALQUALITY RQ-BCR-ABL p190 One-Step kit, code RQ-115, is guaranteed by an
accurate and specific selection of primers and probe and the use of stringent amplification conditions.
Alignment of primers and probes in the most important databanks showed no non-specific pairing.

14.2 Analytical sensitivity: Limit of detection (LoD)

Five points of a serially diluted total RNA extracted from a positive human cell line of BCR-ABLp190
translocation were tested in eight replicates per dilution point in three consecutive analysis sessions.
The detection limit for the REALQUALITY RQ-BCR-ABL p190 One-Step device on the ABI 7500 Fast Dx
system is 1.58 copies of BCR-ABL p190 copies per reaction.

14.3 Analytical sensitivity: linear range

The linear range of this assay was determined using a panel of quantification standards. Analysis of results
was performed using linear regression. The linear range of the REALQUALITY RQ-BCR-ABL p190
One-Step kit on the ABI 7500 Fast Dx system is:

 2.5 to 5 × 106 copies/reaction for BCR-ABL p190, and

 2.5 to 5 × 106 copies/reaction for ABL.

14.4 Reproducibility

In order to determine the assay variability (variability among replicates of the same sample in different
analysis sessions), dilutions of extracted total RNA from positive human cell lines for BCR-ABL p190
translocation e1a2 were tested in five replicates in five consecutive analysis sessions. The variability
coefficient of the device was determined using the cycle threshold value (Ct). The variability coefficient on
the ABI 7500 Fast Dx system is in the range of 0.46 % to 1.39 %.

14.5 Diagnostic specificity

A significant number of samples negative for BCR-ABL p190 were tested simultaneously with the
REALQUALITY RQ-BCR-ABL p190 One-Step kit and another CE IVD device. From the obtained results the
diagnostic specificity of this device on ABI 7500 Fast Dx was calculated to be 100 %.

RQ-BCR-ABL p190_One-Step_manual_e20161209 16
14.6 Diagnostic sensitivity

A significant number of samples positive for BCR-ABL p190 were tested simultaneously with the
REALQUALITY RQ-BCR-ABL p190 One-Step kit and another CE IVD device. From the obtained results the
diagnostic sensitivity of this device on ABI 7500 Fast Dx was calculated to be 100%.

14.7 Accuracy

The accuracy was calculated as the percentage of correct results in relation to the total number of tests. The
total accuracy of REALQUALITY RQ-BCR-ABL p190 One-Step is 100 %.

RQ-BCR-ABL p190_One-Step_manual_e20161209 17
15 REFERENCES
Baccarani M, Saglio G, Goldman J et al. Blood 15;108(6):1809-20, 2006.

Cross N, Müller M, Pane F et al. EUTOS (European Treatment and Outcome Study) for CML. Working
definitions of Molecular Response in CML. Version 4 (August 2014).

Cross N, White H, Colomer D et al. Leukemia 29: 999-1003, 2015.

Gabert J, Beillard E, et al. Leukemia 17(12):2318-57, 2003.

Groffen J, Stevenson JR, Heisterkamp N et al. Cell 36, 93-99, 1984.

Grubbs F, Technometrics 11, 1-21, 1969.

Hochhaus A, Weisser A, La Rosèe P et al. Leukemia 14, 998-1005, 2000.

Labnet, Standard Operating Procedures, v 1.0 del 20-10-2011.

Melo JV. Leukemia 10, 751-756, 1996.

Nowell PC, Hunderford DA. Science 132, 1497, 1960.

Saiki RK, S Scharf, F Faloona, KB Mullis, GT Horn, HA Erlich and N Arnheim, Science 230, 1350-1354,
1985.

Van der Velden VH et al. Leukemia 17, 1013-1034, 2003.

van Dongen JJ et al. Lancet 352, 1731-1738, 1998.

van Dongen JJ, Maclntyre EA, Gabert JA et al. Leukemia12, 1901-1928, 1999.

Verfaillie CM. Biology and therapy of chronic myelogenous leukaemia vol 12, num 1, 1998.

White HE, Matejtschuk P, Rigsby P et al., Blood 116, 111-117, 2010.

White H, Deprez L, Corbisier P et al. Leukemia 1-8, 2014.

15.1 Websites

www.hematology.org
www.bloodjournal.org
www.bloodline.net
www.haematologica.it
www.il-st-acad-sci.org/data6.html
http://medocs.ucdavis.edu/IMD/420A/dib/index.htm
http://web.tiscali.it/ematologia
www.ematologia-italia.net/frame_b.htm
www.simti.it
http://stemcells.alphamedpress.org
www.blacksci.co.uk/uk/society/bsh

RQ-BCR-ABL p190_One-Step_manual_e20161209 18
16 RELATED PRODUCTS
REALQUALITY RQ-BCR-ABL p190 STANDARD
Ready-to-use single-plasmid standards for quantification of the transcripts of BCR-ABL p190 (m-bcr) and
ABL.
This product is in accordance with the 98/79/EC Directive (Annex III) regarding in vitro medical diagnostic
devices (CE marking).

Code Product PKG

RQ-116-SM REALQUALITY RQ-BCR-ABL p190 STANDARD 5 × 110 µL

REALQUALITY RQ-BCR-ABL p210 One-Step

Kit for detection and quantification of translocation t(9;22)(q34;q11) in variant p210 (M-bcr transcripts b3a2
and b2a2) by one-step Real-Time PCR.
This product is in accordance with directive 98/79/EC (Annex III) regarding in vitro medical diagnostic
devices (CE marking).

Code Product PKG

RQ-105-48 REALQUALITY RQ-BCR-ABL p210 One-Step 48 tests
RQ-105-96 REALQUALITY RQ-BCR-ABL p210 One-Step 96 tests

REALQUALITY RQ-BCR-ABL p210 STANDARD

Ready-to-use single-plasmid standards for quantification of the transcripts of BCR-ABL p210 (M-bcr), ABL
and GUSB.
This product is in accordance with the 98/79/EC Directive (Annex III) regarding in vitro medical diagnostic
devices (CE marking).

Code Product PKG

RQ-54-ST REALQUALITY RQ-BCR-ABL p210 STANDARD 5 × 110 µL

BCR-ABL p210 REFERENCE

RNA reference for molecular assays for detection and/or quantification of BCR-ABL p210 transcript.
This product is in accordance with the 98/79/EC Directive (Annex III) regarding in vitro medical diagnostic
devices (CE marking).

Code Product PKG

05-64-06 BCR-ABL p210 REFERENCE 4 × 15 µL

RQ-BCR-ABL p190_One-Step_manual_e20161209 19
17 SYMBOLS

SYMBOL DESCRIPTION

CE mark

Refer to instructions for use

ATTENTION: Refer to accompanying documents

Protect from direct light

Temperature range

Expiration date

Manufacturer

Lot number

Product code

Medical device for in vitro diagnostics

Sufficient for N tests

Limited Licence Agreement

Dye compounds in this product are sold under license from Biosearch Technologies, Inc. and protected by
U.S. and world-wide patents either issued or in application. The license covers Human Molecular Diagnostics
applications.

RQ-BCR-ABL p190_One-Step_manual_e20161209 20