Predicate Devices

Not applicable.

Reference Devices

Not Found

AI/ML (Artificial Intelligence / Machine Learning)

No
The summary describes a standard qPCR assay for quantifying gene transcripts and does not mention any AI or ML components in the device description, intended use, or performance studies. The analysis is based on established molecular biology techniques.

Therapeutic

No
The device is an in vitro diagnostic test for monitoring treatment response in Chronic Myeloid Leukemia patients, not a device that provides therapy.

Diagnostic

No
The "Intended Use" section explicitly states, "This test is not intended for the diagnosis of CML." Instead, it is used for monitoring treatment.

SaMD (Software as a Medical Device)

No

The device description explicitly states it is a "Kit reagents" and includes a table describing the reagents provided, indicating it is a physical kit containing chemical components, not solely software.

IVD (In Vitro Diagnostic)

Yes, this device is an IVD (In Vitro Diagnostic).

Here's why:

Intended Use: The "Intended Use / Indications for Use" section explicitly states that the QuantideX qPCR BCR-ABL IS Kit is an "in vitro nucleic acid amplification test". This is a key characteristic of an IVD.
Sample Type: The test is performed on "total RNA from whole blood", which is a biological sample taken from the human body.
Purpose: The test is intended for the "quantitation of BCR-ABL1 and ABL1 transcripts" and to "measure BCR-ABL1 to ABL1, expressed as a log molecular reduction (MR value)" in CML patients during treatment monitoring. This is a diagnostic purpose, providing information about a patient's health status and response to treatment.
Performance Study: The document describes a clinical performance study using patient samples to evaluate the device's effectiveness in monitoring treatment response. This is typical for IVDs.

The definition of an In Vitro Diagnostic (IVD) is a medical device that is used to examine specimens, such as blood, urine, or tissue, from the human body to detect diseases, conditions, or infections. The QuantideX qPCR BCR-ABL IS Kit fits this definition perfectly.

PCCP (Predetermined Change Control Plan) Authorized

N/A

Overview

Intended Use / Indications for Use

The QuantideX qPCR BCR-ABL IS Kit is an in vitro nucleic acid amplification test for the quantitation of BCR-ABL1 and ABL1 transcripts in total RNA from whole blood of diagnosed t(9,22) positive Chronic Myeloid Leukemia (CML) patients expressing BCR-ABL1 fusion transcripts type e13a2 and/or e14a2. The QuantideX qPCR BCR-ABL IS Kit is a reverse transcription-quantitative PCR performed on the Applied Biosystems 7500 Fast Dx Real-Time PCR Instrument and is intended to measure BCR-ABL1 to ABL1, expressed as a log molecular reduction (MR value) from a baseline of 100% on the International Scale, in t(9;22) positive CML patients during monitoring of treatment with Tyrosine Kinase Inhibitors (TKIs).

The test does not differentiate between e13a2 or e14a2 fusion transcripts and does not monitor other rare fusion transcripts resulting from t(9:22). This test is not intended for the diagnosis of CML.

Product codes (comma separated list FDA assigned to the subject device)

OYX

Device Description

The QuantideX qPCR BCR-ABL IS Kit reagents are adapted for use on the ABI 7500 Fast Dx Real-Time PCR Instrument. The assay includes reagents sufficient for 60 reactions. A description of the reagents provided is described below in Table 1.

Mentions image processing

Not Found

Mentions AI, DNN, or ML

Not Found

Input Imaging Modality

Not Found

Anatomical Site

Not Found

Indicated Patient Age Range

The study included patients with ages ranging from 19 to 75 years, with a mean age of 46.5 years.

Intended User / Care Setting

Not Found

Description of the training set, sample size, data source, and annotation protocol

Not Found

Description of the test set, sample size, data source, and annotation protocol

A retrospective, multi-center clinical outcome study was conducted at 3 US sites to provide evidence of the clinically validity of MR3.0 (0.1% IS) as a threshold for event status at 36 months in CML patients. A total of 139 samples from 98 patients were collected at 2 clinical sites according to protocols approved by the Institutional Review Boards (IRBs). Of those enrolled, a total of 137 evaluable samples were available from 96 subjects. Sample inclusion criteria included the following:

Samples must be in the 12-18 month time frame after the patient started on original or new TKI therapy
Samples from be from adults diagnosed with CML and started with first line or new TKIs
After the date of the sample and through to 32-40 months clinical treatment failure and disease progression status information for event determination is required.

Summary of Performance Studies (study type, sample size, AUC, MRMC, standalone performance, key results)

Clinical Performance Study:

Study Type: Retrospective, multi-center clinical outcome study.
Sample Size: 137 evaluable samples from 96 subjects.
Key Results:
- The device performance was assessed by the probability of at least one event by the endpoint 32-40 months after initiation of TKI treatment as estimated from the Kaplan-Meier survival function.
- Acceptance criteria: probabilities be statistically significantly different and have point estimates differing by at least 10 percentage points.
- The definition of an event included: Death by any cause, The development of accelerated-phase or blast crisis CML, Loss of complete hematologic response, Loss of complete cytogenetic response, Appearance of mutation, Change in TKI treatment not due to toxicity and not due to the results of another BCR-ABL assay.
- EFS estimate difference around MR 3: 22.2% (95% CI: 2.0%-42.4%), p-value 0.0279. This met the acceptance criteria.
- Cox Proportional Hazards (PH) model was used to assess the association of event hazard. Coefficient: -0.642, SE: 0.175, Hazard rate: 0.526, Z: -3.68, P: 0.0002, LR: 14.58 (P=0.0001).
- EFS rates by MR threshold were analyzed. All but the 2 highest thresholds (MR = 4.5) were significant.
- The data support the conclusion that the assay is appropriate for use in BCR-ABL (e13a2 and e14a2) positive CML patients during monitoring of treatment with Tyrosine Kinase Inhibitors (TKIs) around a threshold of MR3 (the pre-specified threshold for clinical use).

Analytical Performance Studies:

Precision:
- Within-laboratory Precision: Five human RNA specimens positive for BCR-ABL1 were diluted spanning MR 1.0 to MR 4.0. Testing spanned 3 lots, 3 operators, 20 runs, and 3 instruments. The maximum observed standard deviation was 0.134. Acceptance criteria were met.
- Site-to-Site Reproducibility: The same 25 sample pool was evaluated at 4 sites by multiple operators over multiple nonconsecutive days for a total of 1200 measurements. Acceptance criteria were met.
Linearity/Reportable Range:
- Two separate RNA specimens (e13a2 and e14a2) were diluted from MR 0.1 to MR 4.8. Testing across 2 lots.
- Linear regression curves demonstrated slopes of 1.01 for both transcripts.
- Transcript e13a2 was linear from MR 0.12 to MR 4.84 with a maximum SD of 0.17.
- Transcript e14a2 was linear from MR 0-.22 to MR 4.78 with a maximum SD of 0.17.
- Supports linearity from at least MR 0.3 (50% IS) to MR 4.7 (0.002% IS).
Detection Limit:
- Limit of Blank (LoB): Determined by testing 30 non-leukemic human RNA specimens. 263 out of 265 valid measurements were undetectable.
- Limit of Detection (LoD): Determined using 4 separate human RNA specimens serially diluted. LoD for each transcript was 0.002% IS/MR 4.7.
- Limit of Quantitation (LoQ): Determined by testing 6 specimens targeting MR 4.7. MR values ranged from 4.6 to 4.87 with SD values from 0.23 to 0.34. LoQ is equivalent to LoD (MR 4.7).
Analytical Specificity:
- Interfering Substances: Tested with substances at the LoQ. Acceptance criteria were met, none interfered at tested concentrations.
- Primer Specificity: Tested using 11 leukemic and 2 non-leukemic RNA specimens. Acceptance criteria were met, indicating exclusive detection of BCR-ABL1 e13a2 and e14a2 fusion transcripts.
- Specimen Carryover Contamination: Tested by alternating high positive (MR 0.8) and CML-negative specimens. All negative specimens (25/25) were reported as "Negative (sufficient ABL1)". Acceptance criteria were met.
RNA Isolation and RNA Input:
- RNA Isolation: Tested with 3 commonly used RNA extraction methods. All methods gave equivalent results.
- RNA Input: Tested targeted MR values of 1, 3, or 4 with target RNA inputs ranging from 250 ng to 6000 ng. Acceptance criteria were met for RNA inputs from 750 to 6000 ng from MR 1 to MR 4.5, supporting the recommended range of 1000-5000 ng.
Traceability and Stability:
- Traceability: Calibrators are traceable to the First WHO International Genetic Reference Panel. Demonstrated by measuring the WHO Reference Panel with 3 assay kit lots. Acceptance criteria were met (slope close to 1, intercept close to 0).
- Reagent Stability:
  - Real-time Stability: Ongoing testing at various time points (T0, T3, T6, T9, T12, T13 months). Current stability is 6 months.
  - Freeze-thaw Stability: Stable up to 4 uses (including freeze-thaw between uses).
  - Shipping Stability: Stable following shipment of up to 72 hrs on dry ice under specified stress conditions.
- Specimen Stability (Whole Blood Stability): Tested CML positive clinical whole blood specimens stored at 2-8°C for up to 72 hrs. Acceptance criteria were met.

Key Metrics (Sensitivity, Specificity, PPV, NPV, etc.)

EFS Estimate Difference: 22.2% (95% CI: 2.0%-42.4%), p-value 0.0279 when comparing MR = 3.
Cox PH Model: Hazard rate 0.526, P=0.0002.

Predicate Device(s): If the device was cleared using the 510(k) pathway, identify the Predicate Device(s) K/DEN number used to claim substantial equivalence and list them here in a comma separated list exactly as they appear in the text. List the primary predicate first in the list.

Not applicable.

Reference Device(s): Identify the Reference Device(s) K/DEN number and list them here in a comma separated list exactly as they appear in the text.

Not Found

Predetermined Change Control Plan (PCCP) - All Relevant Information for the subject device only (e.g. presence / absence, what scope was granted / cleared under the PCCP, any restrictions, etc).

Not Found

Regulation Number and Section

§ 866.6060 BCR-ABL quantitation test.

(a)
Identification. A BCR-ABL quantitation test is identified as a reverse transcription-quantitative polymerase chain reaction (RT-qPCR) test for the quantitation of BCR-ABL1 expressed on the International Scale (IS) and control transcripts in total RNA from whole blood of diagnosed t(9;22) positive chronic myeloid leukemia (CML) patients during monitoring of treatment with tyrosine kinase inhibitors. This test is not intended for the diagnosis of CML.(b)
Classification. Class II (special controls). The special controls for this device are:(1) Premarket notification submissions must include the following information:
(i) The indication for use must indicate the variant(s) for which the assay was designed and validated, for example BCR-ABL e13a2 and/or e14a2.
(ii) A detailed description of all components in the test, including the following:
(A) A detailed description of the test components, all required reagents, instrumentation and equipment, including illustrations or photographs of non-standard equipment or methods;
(B) Detailed documentation of the device software including, but not limited to, standalone software applications and hardware-based devices that incorporate software;
(C) Methodology and protocols for control procedures for the assay to allow reporting on the International Scale;
(D) A description of the result outputs, analytical sensitivity of the assay, and the range of values that will be reported; and
(E) A description of appropriate internal and external controls that are recommended or provided. The description must identify those control elements that are incorporated into the testing procedure.
(iii) Information that demonstrates the performance characteristics of the test, including:
(A) For indications for use based on a threshold established in a predicate device of this generic type, device performance data from either a method comparison study to the predicate device or through a clinical study demonstrating clinical validity using well-characterized prospectively or retrospectively obtained clinical specimens, as appropriate, representative of the intended use population;
(B) For indications for use based on a threshold not established in a predicate device of this generic type, device performance data from a clinical study demonstrating clinical validity using well-characterized prospectively or retrospectively obtained clinical specimens, as appropriate, representative of the intended use population;
(C) Device reproducibility data generated, using a minimum of three sites, of which at least two sites must be external sites, with two operators at each site. Each site must conduct a minimum of three runs per operator over non-consecutive days evaluating a minimum of five different BCR-ABL concentrations that span and are well distributed over the measuring range and include MR3 (0.1 percent IS). Results shall be reported as the standard deviation and percentage coefficient of variation for each level tested. Prespecified acceptance criteria must be provided and followed;
(D) Device precision data using clinical samples to evaluate the within-lot, between-lot, within-run, between run, and total variation;
(E) Device linearity data using a dilution panel created from clinical samples;
(F) Device analytic sensitivity data, including limit of blank, limit of detection, and limit of quantification;
(G) Device specificity data, including interference and cross-contamination; and
(H) Device stability data, including real-time stability of samples under various storage times, temperatures, and freeze-thaw conditions.
(iv) Identification of risk mitigation elements used by your device, including a detailed description of all additional procedures, methods, and practices incorporated into the instructions for use that mitigate risks associated with testing using your device.
(2) Your 21 CFR 809.10 compliant labeling must include the following:
(i) The intended use in your 21 CFR 809.10(a)(2) and (b)(2) complaint labeling must include an indication for use statement that reads “This test is not intended for the diagnosis of CML”; and
(ii) A detailed description of the performance studies conducted to comply with paragraph (b)(1)(iii) of this section and a summary of the results.
(3) Your device output must include results on the International Scale (IS) and your assay must include multipoint calibration controls traceable to a relevant international reference panel (
e.g., the World Health Organization International Genetic Reference Panel for quantitation of BCR-ABL mRNA).

Summary

0

EVALUATION OF AUTOMATIC CLASS III DESIGNATION FOR QUANTIDEX QPCR BCR-ABL IS KIT DECISION SUMMARY

A. DEN Number:

DEN160003

B. Purpose for Submission:

De novo request for evaluation of automatic class III designation of the QuantideX qPCR BCR-ABL IS Kit

C. Measurand:

BCR-ABL1 and ABL1 transcripts

D. Type of Test:

Reverse transcription, quantitative, polymerase chain reaction (qPCR) based nucleic acid amplification

E. Applicant:

Asuragen

F. Proprietary and Established Names:

Trade Name: QuantideX qPCR BCR-ABL IS Kit Common Name: BCR-ABL1 RT-qPCR Test

G. Regulatory Information:

1. Regulation section:
21 CFR 866.6060
1. Classification:

Class II (Special Controls)

1. Product code:
  OYX
1. Panel:
88 Pathology

1

H. Indications For Use:

1. Indications for Use

The QuantideX qPCR BCR-ABL IS Kit is an in vitro nucleic acid amplification test for the quantitation of BCR-ABL1 and ABL1 transcripts in total RNA from whole blood of diagnosed t(9,22) positive Chronic Myeloid Leukemia (CML) patients expressing BCR-ABL1 fusion transcripts type e13a2 and/or e14a2. The QuantideX qPCR BCR-ABL IS Kit is a reverse transcription-quantitative PCR performed on the Applied Biosystems 7500 Fast Dx Real-Time PCR Instrument and is intended to measure BCR-ABL1 to ABL1, expressed as a log molecular reduction (MR value) from a baseline of 100% on the International Scale, in t(9;22) positive CML patients during monitoring of treatment with Tyrosine Kinase Inhibitors (TKIs).

The test does not differentiate between e13a2 or e14a2 fusion transcripts and does not monitor other rare fusion transcripts resulting from t(9:22). This test is not intended for the diagnosis of CML.

1. Special conditions for use statement(s):
  For in vitro diagnostic use only.

For prescription use only.

1. Special instrument requirements:
  Applied Biosystems 7500 Fast Dx Real-Time PCR Instrument using System Sequence Detection Software v1.4.1.

I. Device Description:

The QuantideX qPCR BCR-ABL IS Kit reagents are adapted for use on the ABI 7500 Fast Dx Real-Time PCR Instrument. The assay includes reagents sufficient for 60 reactions. A description of the reagents provided is described below in Table 1.

Item	Description	Use
QuantideX qPCR BCR-ABL IS Reagents (Box 1 of 2)
BCR-ABL IS Diluent	Nuclease-free water	Adjust volume of RT reactions
BCR-ABL IS RT Buffer	Deoxynucleotides (dNTPs) and degenerate
deoxyoligonucleotides in a
buffered salt solution	Random priming of RNA templates and primer extension
to generate cDNA from RNA
templates
BCR-ABL IS RT Enzyme Mix	Reverse transcriptase	Enzyme component of the RT
reaction to generate cDNA from
RNA template
BCR-ABL IS qPCR Buffer	Deoxynucleotides (dNTPs) in a
buffered salt solution	Provides dNTPs for primer
extension to generate DNA
amplicons from cDNA
templates
BCR-ABL IS Primer Probe Mix	Deoxyoligonucleotide primers, dye- and quencher- conjugated deoxyoligonucleotide probes, and passive reference dye	Provides primers and probes for PCR amplification and detection of target sequences. Passive reference dye is used by the instrument software to normalize fluorescence throughout the PCR
BCR-ABL IS Enzyme Mix	Thermostable DNA polymerase	Catalyzes the amplification of templates hybridized to templates in the cDNA. Enzyme exonuclease activity degrades hybridized probes to release fluorescence for detection of amplicons in each PCR cycle.
QuantideX qPCR BCR-ABL IS Calibrators and Controls (Box 2 of 2)
BCR-ABL IS CAL1	BCR-ABL and ABL armored RNA formulated to approximately MR 1.0	Generate calibration curve for calculation of MR values for controls and patients specimens
BCR-ABL IS CAL2	BCR-ABL and ABL armored RNA formulated to approximately MR 2.0
BCR-ABL IS CAL3	BCR-ABL and ABL armored RNA formulated to approximately MR 3.0
BCR-ABL IS CAL4	BCR-ABL and ABL armored RNA formulated to approximately MR 4.0
BCR-ABL IS CONH	BCR-ABL and ABL armored RNA formulated to approximately MR 1.5	Control used to ensure that RT and PCR steps performed properly by generating expected MR value
BCR-ABL IS CONL	BCR-ABL and ABL armored RNA formulated to approximately MR 3.5	Control used to ensure that RT and PCR steps performed properly and protect against contamination and falsely positive samples to due contamination by generating a results of "Negative (Sufficient ABL1)"
BCR-ABL IS CONN	ABL armored RNA

Table 1: Components of the QuantideX Assay

2

Additional materials required but not provided with the QuantideX qPCR BCR-ABL IS Kit:

1. Reagents for total RNA isolation
1. Applied Biosystems 7500 Fast Dx Real-Time PCR instrument

J. Substantial Equivalence Information:

1. Predicate device name(s) and DEN number(s): Not applicable.

3

1. Comparison with predicate: Not applicable.

K. Standards/Guidance Documents Referenced:

Guidance for Industry and FDA Staff: Guidance for the Content of Premarket Submission for . Software Contained in Medical Devices
General Principles of Software Validation; Final Guidance for Industry and FDA Staff .
. Guidance for Industry, FDA Reviewers and Compliance on Off-the-Shelf Software Use in Medical Devices
CLSI EP05-A3, Evaluation of Precision Performance of Quantitative Measurement Methods
CLSI EP06-A, Evaluation of the Linearity of Quantitative Measurement Procedures, A ● Statistical Approach
CLSI EP07-A2, Interference Testing in Clinical Chemistry ●
CLSI EP25-A. Evaluation of Stability on In Vitro Diagnostic Reagents .

L. Test Principle:

The QuantideX qPCR BCR-ABL IS Kit is a nucleic acid amplification test for the quantitation of BCR-ABL1 RNA. The assay provides simultaneous amplification and detection of two BCR-ABL1 fusion transcripts (e13a2 and e14a2) and ABL1 (an endogenous control) using total RNA extracted from human white blood cells enriched from EDTA whole blood. The test uses multiplex reverse transcription-PCR (RT-PCR) in combination with real-time hydrolysis probe technology. BCR-ABL1 translocations and ABL1 mRNA are simultaneously reverse-transcribed, amplified, detected, and quantified in a single reaction well. b(4)

This design is diagrammed below.

Figure 1: Primer and Probe Design for the QuantideX Assay b(4)

4

The RNA quantity of BCR-ABL is assessed relative to ABL1 expression within each reaction well, and is computed two ways: (1) as the specimen percent IS value (the percent ratio of BCR-ABLI to ABL1 expressed on the International Scale), and (2) as a molecular reduction value (MR, a logarithmic decrease from the common baseline of 100% IS or MR 0). ABL1 also serves as a control for assessment of RNA quality for the specimen.

The Assay Calibrator set includes external calibrators built with Armored RNA® Quant (ARQ) technology to generate traceable %IS values (transformed into the primary measurement of a molecular reduction (MR) from the common baseline of 100%IS) from a single calibration curve (provided). Calibrators are run in duplicate and all other specimens and controls may be run in singlicate. The time required from the RT set-up step through the generation of the last test result is $\n[ \leq ]\n$ 4 hours for a full kit.

M. Interpretation of Results

The numerical value of the World Health Organization (WHO) International Scale is % IS, the ratio expressed as a percentage of BCR-ABL1 expression to the expression of a control gene (ABL1 in this instance). The International Scale (%IS) is a geometric progression and therefore repeated measurements of a sample are non-normally distributed about the mean. %IS values require logtransformation prior to performing any statistical analyses that require normally-distributed data.

Another value commonly reported in the literature is the Molecular Reduction, or MR value. The MR value is traditionally written as $MR^{x.x}$. However, for simplicity and legibility, the QuantideX assay will report the value as MRx.x. The MR value is the logio reduction from the internationally standardized baseline, defined as 100% IS. Therefore,

$$MR\ge\colon \pi = \log_{10}\left(\frac{100}{% \delta IS}\right) = \log_{10}\left(100\right) - \log_{10}\left(% \delta IS\right) = 2 - \log_{10}\left(% \delta IS\right)$$

The test uses MR values for the calibration standards as well as the primary specimen output, with %IS also reported. MR values with their corresponding %IS values are shown below in Table 2.

MR	%IS
0.0	100
0.5	32
1.0	10
1.5	3.2
2.0	1
2.5	0.32
3.0	0.1
3.5	0.032
4.0	0.01
4.5	0.0032
4.7	0.002
5.0	0.001

Table 2: MR Values and Corresponding %IS Values

N. Performance Characteristics:

5

1. Analytical Performance:

a. Precision:
- i. Within-laboratory Precision: Five human RNA specimens positive for BCR-ABL1 were diluted into RNAs from human CML-negative blood to formulate 25 samples ranging from MR 1.0 to MR 4.0, encompassing both transcript types (e13a2 and e14a2). Testing spanned 3 lots, 3 operators, 20 runs, and 3 instruments. The acceptance criteria for this study were that the standard deviations satisfy the criteria in Table 3. The results of the precision study are shown in Table 4. The range of observed standard deviations at each MR level across the 5 specimens is summarized in Table 5. The maximum observed standard deviation was 0.134. The acceptance criteria for this study were met supporting the conclusion that the assay has acceptable repeatability 0.0316 | ≤ 50 |
  | 3.5-4.25 | ≤0.29 | 0.0316-0.0056 | ≤ 75 |
  | > 4.25 | ≤0.36 | MR 1.0 $\n[ \leq ]\n$ 4.0.

| Targeted
MR | Sample | Site
SD | Day
SD | Operator
SD | Within
SD | Total
SD |
|----------------|--------|------------|-----------|----------------|--------------|-------------|
| MR1 | pAls01 | 0.008 | 0.000 | 0.012 | 0.028 | 0.049 |
| | pAls06 | 0.009 | 0.014 | 0.000 | 0.021 | 0.044 |
| | pAls11 | 0.024 | 0.017 | 0.014 | 0.022 | 0.077 |
| | pAls16 | 0.017 | 0.020 | 0.000 | 0.019 | 0.056 |
| | pAls21 | 0.013 | 0.021 | 0.011 | 0.020 | 0.064 |
| MR2 | pAls02 | 0.000 | 0.021 | 0.000 | 0.068 | 0.089 |
| | pAls07 | 0.000 | 0.022 | 0.004 | 0.017 | 0.044 |
| | pAls12 | 0.032 | 0.009 | 0.026 | 0.021 | 0.087 |
| | pAls17 | 0.012 | 0.016 | 0.009 | 0.019 | 0.055 |
| | pAls22 | 0.012 | 0.014 | 0.000 | 0.031 | 0.057 |
| MR3 | pAls03 | 0.039 | 0.024 | 0.005 | 0.036 | 0.104 |
| | pAls08 | 0.000 | 0.023 | 0.009 | 0.028 | 0.059 |
| | pAls13 | 0.039 | 0.026 | 0.000 | 0.052 | 0.117 |
| | pAls18 | 0.018 | 0.000 | 0.009 | 0.033 | 0.059 |

Table 6: Summarized Variance for all Samples and Pools in Multi-Site Reproducibility

7

MR3.5	pA1s23	0.036	0.019	0.000	0.032	0.087
	pA1s04	0.061	0.023	0.026	0.037	0.147
	pA1s09	0.013	0.042	0.010	0.043	0.108
	pA1s14	0.038	0.039	0.041	0.051	0.169
	pA1s19	0.026	0.010	0.000	0.035	0.071
	pA1s24	0.050	0.027	0.000	0.032	0.109
MR4	pA1s05	0.054	0.027	0.032	0.053	0.167
	pA1s10	0.029	0.000	0.000	0.067	0.095
	pA1s15	0.014	0.037	0.000	0.070	0.122
	pA1s20	0.042	0.000	0.000	0.079	0.121
	pA1s25	0.026	0.028	0.005	0.063	0.123

b.Linearity/Reportable Range:

Linearity was estimated by testing 2 separate RNA specimens that were positive for BCR-ABL (one e13a2 and one e14a2). Because an appropriate reference method could not be identified, transcript quantity is relative to the QuantideX assay. Each was diluted into RNAs from CML-negative whole blood to a range of MR 0.1 to MR 4.8. Testing was conducted across 2 lots of assay kits. The acceptance criteria for this testing were the same as those described in Table 3 above.

The linear regression curves demonstrated slopes of 1.01 for both transcripts and intercepts of -0.11 and -0.05 for e13a2 and e14a2, respectively. Transcript e13a2 was linear from MR 0.12 to MR 4.84 with a maximum SD of 0.17 and transcript e14a2 was linear from MR 0-.22 to MR 4.78 with a maximum SD of 0.17.

Second and 3th order polynomial regressions were also assessed and the analysis supports linearity from at least MR 0.3 (50% IS) to MR 4.7 (0.002% IS). These results support the conclusion that the assay is linear for both transcripts from MR 0.3 (50%IS) to MR4.7 (0.002%IS).

8

Image /page/8/Figure/0 description: The image contains two scatter plots, labeled 'e13a2' and 'e14a2'. Both plots show a strong positive correlation between 'Targeted MR' on the x-axis and 'MR' on the y-axis. The data points are clustered tightly around a diagonal line, indicating a close agreement between the targeted and actual MR values, ranging from 0 to 5 on both axes.

Figure 2: Linearity of e13a2 (left) and e14a2 (right) transcripts

c. Detection Limit:

i. Limit of Blank (LoB):
The LoB was determined by testing 30 non-leukemic human RNA specimens that were presumed negative for BCR-ABL. Testing spanned 3 assay kit lots, 4 operators, 9 runs, 4 calendar days and 4 qPCR instruments for a total of 265 valid measurements.

Out of 265 valid measurements, 263 were undetectable.

ii. Limit of Detection (LoD):
The LoD was determined using 4 separate human RNA specimens that were positive for BCR-ABL1, each serially diluted into RNAs from human CML-negative whole blood to 28 dilution levels. Testing spanned 2 assay kit lots, 4 operators, 15 calendar days, and 4 qPCR instruments. Both e13a2 and e14a2 transcripts were evaluated.

The non-parametric method to determine LoD for quantitative devices was used, keeping the type II error under 5%. The median value of the tested %IS values across all included panel member replicates was determined and defined as the LoD. This analysis vielded a LoD for each transcript of 0.002% IS/MR 4.7. The results are pictured below in Table 7.

| Transcript | Specimens | Total
Results | Total
Positive | Percent
Undetected | Median
%IS | LOD
(MR) |
|------------|--------------------------------------------------------------|------------------|-------------------|-----------------------|---------------|-------------|
| e13a2 | pC1s02, pC1s24, pC1s25 | 179 | 172 | 3.9 | 0.002 | 4.70 |
| e14a2 | pC1s09, pC1s10, pC1s11,
pC1s12, pC1s17, pC1s18,
pC1s19 | 420 | 400 | 4.8 | 0.002 | 4.70 |

Table 7: Determination of LoD for both BCR-ABL Transcripts

9

iii. Limit of Quantitation (LoO):

The LoQ was determined by testing 6 specimens that were derived from 6 human RNA that were positive for BCR-ABL (each diluted into RNA from human CML-negative whole blood to the target of MR 4.7).The results were evaluated against the acceptance criteria of SD ≤ 0.36 at MR 4.5 or greater. Testing generated 120 measurements across 2 lots of kit. MR values ranged from 4.6 to 4.87 with SD values ranging from 0.23 to 0.34. The results indicate that the LoQ is equivalent to the LoD (MR 4.7).

d.Analytical Specificity:

i. Interfering Substances:
Potential interfering substances from both blood sources and RNA extraction sources were tested with specimens at the LoQ in the following concentrations:
Hemoglobin 200 g/L
Lipid 5.6 mM ●
Albumin 50 g/L
Conjugated bilirubin 86 mM
Unconjugated bilirubin 257 µM ●
Guanidinium containing lysis buffer 1%
Ethanol 7% ●
Phenol 0.1%
Assay wash buffer 10%
Genomic DNA 50 ng/RT ●

The RNA specimens tested were residual CML-positive blood diluted to approximately MR4.0 into RNA from CML-negative whole blood. Testing was performed in 9 replicates and compared to control samples. The acceptance criteria were that the mean of the test samples was ± 0.5 MR of the control. The acceptance criteria were met, supporting the conclusion that none of the tested agents interfered with the assay at the concentrations listed.

ii. Primer Specificity:
Analytical specificity (i.e., exclusivity) for the BCR-ABL breakpoints e13a2 and e14a2 was tested using 11 leukemic specimens (CML, Acute Myeloid Leukemia (AML), Acute Lymphoblastic Leukemia (ALL)) and 2 non-leukemic RNA specimens. Testing generated 117 measurements across 3 lots of assay kit. The acceptance criteria were met in that specimens without a major BCR-ABL1 break point were reported as negative for BCR-ABL1 or below LoD in > 8/9 replicates. The results across the various samples are shown in Table 8. The results support supported the conclusion that the test detects the BCR-ABL1 e13a2 and e14a2 fusion transcripts exclusively.

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| Leukemia

Type	Fusion Transcript	Sample type	Result2
CML or ALL	t(9;22)
minor breakpoint e1a2 (p190)	Cell line RNA	95% of CML- negative samples tested negative or below LoD.

Testing found that all negative specimens (25/25) were reported as "Negative (sufficient ABL1)". All high positive samples gave an appropriate MR value (mean MR value of 0.79 [16%IS], range of MR0.73 to 0.83 [19 to 15%IS, respectively]). The acceptance criteria were met supporting the conclusion that the test generates no significant carryover between wells on the plate.

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e.RNA Isolation and RNA Input:

i. RNA Isolation:

Three commonly used RNA extraction methods were tested using CML-positive white blood cells serially diluted across 4 levels (MR 1-4) into CML-negative human anti-coagulated whole blood. Testing incorporated both transcripts (e13a2 and e14a2). Two lots of assay kits were used in this testing. The acceptance criteria were that each MR value would conform to the SD criteria described in Table 3 above.

All 3 isolation methods gave equivalent results with the standard deviation within each method being less than 0.1 for all levels and lots. Therefore, it is concluded that the test is compatible with generic methods of RNA isolation, and any validated method of RNA extraction and isolation that yields unbiased isolation of total RNA in sufficient quantity and quality may be used with the assay.

ii. RNA Input:

The assay uses an RNA input concentration of 1000 to 5000 ng. To validate that the performance of the assay across the RNA concentrations, an RNA input study was conducted. A total of 30 samples derived from 2 primary RNA samples diluted into nonleukemic human RNA were tested. Samples were set to target MR values of 1, 3, or 4 with target RNA inputs ranging from 250 ng to 6000 ng, and were run in 9 replicates per sample giving a total of 216 evaluable observations. Acceptance criteria were the same as those described in Table 3 above. The results are shown in Table 9 below. The acceptance criteria were met for RNA inputs ranging from 750 to 6000 ng from MR 1 to MR 4.5, supporting the recommended RNA input range of 1000-5000 ng.

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| Dilution
Series | Specimen | RNA
Input
(ng) | Reps | Percent
Positive | Mean
MR | SD
(MR) | Mean
%IS | %CV
(%IS) | Series
Mean
MR | Series
SD
(MR) | Batch
Mean
%IS | Batch
%CV
(%IS) |
|--------------------|----------|----- | 1 | pR1s01 | 6000 | | pR1s02 | 5000 | | pR1s03 | 3000 | | pR1s04 | 1000 | | pR1s05 | 750 | | pR1s06 | 250 | 2 | pR1s07 | 6000 | | pR1s08 | 5000 | | pR1s09 | 3000 | | pR1s10 | 1000 | | pR1s11 | 750 | | pR1s12 | 250 | 3 | pR1s13 | 6000 | | pR1s14 | 5000 | | pR1s15 | 3000 | | pR1s16 | 1000 | | pR1s17 | 750 | | pR1s18 | 250 | 5 | pR2s01 | 6000 | | pR2s02 | 5000 | | pR2s03 | 3000 | | pR2s04 | 1000 | | pR2s05 | 750 | | pR2s06 | 250 -----------------|------|---------------------|------------|------------|-------------|--------------|----------------------|----------------------|----------------------|-----------------------|
| 9 | 100 | 0.99 | 0.06 | 10.2263 | 13.3 | 1.00 | 0.04 | 10.1028 | 9.0 |
| 9 | 100 | 1.00 | 0.03 | 10.0471 | 7.9 | | | | |
| 9 | 100 | 1.00 | 0.04 | 10.0171 | 9.7 | | | | |
| 9 | 100 | 0.98 | 0.02 | 10.5391 | 5.7 | | | | |
| 9 | 100 | 1.00 | 0.05 | 10.0122 | 10.4 | | | | |
| 9 | 100 | 1.01 | 0.02 | 9.7939 | 5.1 | | | | |
| 9 | 100 | 3.05 | 0.06 | 0.0905 | 13.6 | 3.06 | 0.07 | 0.0886 | 16.0 |
| 9 | 100 | 3.08 | 0.06 | 0.0844 | 13.5 | | | | |
| 9 | 100 | 3.07 | 0.03 | 0.0859 | 6.8 | | | | |
| 9 | 100 | 3.05 | 0.03 | 0.0897 | 7.8 | | | | |
| 9 | 100 | 3.06 | 0.08 | 0.0892 | 17.3 | | | | |
| 9 | 100 | 3.05 | 0.13 | 0.0928 | 30.0 | | | | |
| 9 | 100 | 4.67 | 0.23 | 0.0025 | 55.7 | 4.70 | 0.27 | 0.0024 | 68.4 |
| 9 | 100 | 4.69 | 0.27 | 0.0024 | 69.7 | | | | |
| 9 | 100 | 4.73 | 0.29 | 0.0023 | 72.5 | | | | |
| 9 | 89 | 4.67 | 0.32 | 0.0028 | 84.3 | | | | |
| 9 | 89 | 4.75 | 0.22 | 0.0020 | 54.1 | | | | |
| 9 | 33 | 4.66 | 0.52 | 0.0044 | 173.1 | | | | |
| 9 | 100 | 4.30 | 0.09 | 0.0051 | 19.8 | 4.28 | 0.21 | 0.0059 | 50.6 |
| 9 | 100 | 4.32 | 0.21 | 0.0053 | 53.0 | | | | |
| 9 | 100 | 4.27 | 0.15 | 0.0057 | 35.5 | | | | |
| 9 | 100 | 4.32 | 0.29 | 0.0060 | 75.4 | | | | |
| 9 | 100 | 4.36 | 0.17 | 0.0047 | 40.2 | | | | |
| 9 | 100 | 4.11 | 0.22 | 0.0089 | 54.7 | | | | |

Table 9: RNA Input Study Results

f. Traceability and Stability (Reagent and Specimen):

i. Traceability:

The assay calibrators are traceable to the First (1ªt) WHO International Genetic Reference Panel. White HE, Matejtschuk P, Rigsby P, et al. Establishment of the first World Health Organization International Genetic Reference Panel for quantitation of BCR-ABL mRNA. Blood. 2010;116(22):e111-7. The Value Assignment process for the calibrators is depicted below in Figure 3. Traceability to the 1st WHO International Genetic Reference Panel for quantitation of BCR-ABL translocation by RQ-PCR was demonstrated by measuring the WHO Reference Panel with 3 assay kit lots and comparing the measured values to the published values of the panel. Each of the 4 panel members was tested in duplicate across 3 runs (1 run per lot). The MR values for each level of the reference panel were calculated by regression to each lot of the kit calibrator. Measured MR values were compared to published MR values through an additional

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regression analysis to determine the slope and intercept. The acceptance criteria for this testing were that the regression model have a slope close to 1 (1.0 to 1.1.) and an intercept close to 0 (0.02-0.11). The results are pictured below in Figure 4. The acceptance criteria were met supporting the conclusion that traceability of the assay calibrators to the WHO Reference Panel has been established.

Image /page/13/Figure/1 description: The image is a title for a figure. The title reads "Figure 3: Depiction of the Traceability of the QuantideX Calibrators to the WHO Reference Standard Panel." The title is written in a clear, sans-serif font and is centered on the page. The text provides context for the figure.

Image /page/13/Figure/2 description: The image shows a diagram of the WHO IS Panel (1RM), Asuragen Secondary Reference (2RM), and QuantideX Kit Calibrators (3RM). The WHO IS Panel (1RM) includes an international standard, cell line blends, limited supply, and values assigned by international consensus. The Asuragen Secondary Reference (2RM) is used to assign IS values to each lot of kit calibrators and has an unlimited supply. The QuantideX Kit Calibrators (3RM) include routine lots each aligned to WHO Primary using 2RM.

Figure 4: Measured vs. Published Values for WHO Standards by Lot

Image /page/13/Figure/4 description: The image shows three scatter plots comparing Kit-determined MR values to published MR values for WHO Primary, separated by LOT number. Each plot displays a linear regression line with its equation and R-squared value. For LOT1, the equation is y = -0.11 + 1.1x with r^2 = 0.995; for LOT2, y = 0.02 + 1x with r^2 = 0.996; and for LOT3, y = -0.059 + 1.1x with r^2 = 0.999. The data points for each LOT are represented by different colors: LOT1 in black, LOT2 in blue, and LOT3 in orange.

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ii. Reagent Stability:

Real-time Stability: a.
Reagent shelf life stability studies were conducted using 3 lots with testing ongoing at T0, T3, T6, T9, T12, and T13 months. At each time point 15 replicates are tested (5 replicates/lot). Samples are total RNA derived from residual CML positive whole blood serially diluted into total RNA from human CML negative whole blood. The 5 samples are diluted to approximately MR 1, 2, 3, 4, and