Predicate Devices

Reference Devices

AI/ML (Artificial Intelligence / Machine Learning)

No
The summary describes a quantitative PCR system and associated software for analyzing genetic material. There is no mention of AI or ML in the device description, intended use, or performance studies. The analysis is based on counting positive and negative droplets, which is a direct measurement technique, not an AI/ML inference process.

Therapeutic

No
The device is an in vitro diagnostic (IVD) test used to quantify BCR-ABL1 and ABL1 transcripts for monitoring CML patients, not to provide therapy.

Diagnostic

Yes

This device is designed for the quantitation of BCR-ABL1 and ABL1 transcripts in blood, which is used to monitor treatment efficacy in CML patients. While stated it "is not intended for the diagnosis of CML" itself, the quantitation and monitoring of disease markers for treatment management is a diagnostic function.

SaMD (Software as a Medical Device)

No

The device description explicitly states that the system consists of two instruments (QXDx Automated Droplet Generator and QXDx Droplet Reader) and associated consumables, in addition to the software. This indicates the presence of significant hardware components essential to the device's function.

IVD (In Vitro Diagnostic)

Based on the provided information, the QXDx™ BCR-ABL %IS Kit is indeed an IVD (In Vitro Diagnostic).

Here's why:

Intended Use: The "Intended Use / Indications for Use" section explicitly states that the kit is an "in vitro nucleic acid amplification test". This is a key characteristic of an IVD. It is designed to be used outside of the body to examine samples (in this case, total RNA from whole blood) for diagnostic or monitoring purposes.
Purpose: The kit is intended for the quantitation of BCR-ABL1 and ABL1 transcripts in blood samples from diagnosed CML patients. This measurement is used for monitoring treatment with Tyrosine Kinase Inhibitors (TKIs) and expressing the results on the International Scale. This is a clear diagnostic/monitoring purpose.
Sample Type: The test is performed on total RNA from whole blood, which is a biological sample collected from a patient.
Device Description: The description details the components of the kit and the associated system (QXDx AutoDG ddPCR System) used to perform the test. These are the tools and reagents used in the in vitro diagnostic process.
Performance Studies: The document includes detailed descriptions of Analytical Performance and Clinical Performance (Method Comparison) studies. These studies are conducted to demonstrate the accuracy, precision, and reliability of the test for its intended diagnostic/monitoring use.
Predicate Device(s): The mention of "Predicate Device(s)" with K numbers (De Novo DEN160003 QuantideX qPCR BCR-ABL IS Kit; Class II K141220 Applied Biosystems 7500 Fast Dx Real-Time PCR Instrument with SDS Software) indicates that this device is being compared to or is similar to other devices that have gone through regulatory review as IVDs.

In summary, the QXDx™ BCR-ABL %IS Kit meets the definition of an In Vitro Diagnostic device because it is a test performed outside the body on biological samples to provide information for the diagnosis, monitoring, or treatment of a disease (Chronic Myeloid Leukemia in this case).

PCCP (Predetermined Change Control Plan) Authorized

N/A

Overview

Intended Use / Indications for Use

The QXDx™ 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 QXDx BCR-ABL %IS Kit is a reverse transcription-quantitative PCR performed on the Bio-Rad QXDx™ AutoDG™ ddPCR System 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 QXDx BCR-ABL %IS Kit is intended for use only on the Bio-Rad QXDx AutoDG ddPCR System.

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

OYX, PHG

Device Description

The QXDx AutoDG ddPCR System consists of two instruments, the QXDx Automated Droplet Generator and the QXDx Droplet Reader, and their associated consumables. The QXDx Automated Droplet Generator partitions samples into approximately 20,000 nanoliter-sized droplets and, after PCR on a thermal cycler, droplets from each sample are analyzed individually on the QXDx Droplet Reader. PCR-positive and PCR-negative droplets are counted to provide direct quantification of nucleic acid in digital form. Results are analyzed on QXDx Software running on a Windows based computer.

The QXDx AutoDG ddPCR System contains:

QXDx Automated Droplet Generator
QXDx Droplet Reader
Laptop Computer with QXDx Software
Accessory components:
- ddPCR Dx AutoDG Consumable Pack
  - Automated Droplet Generation Oil for Probes
  - DG32 Cartridges w/ Gaskets
  - ddPCR Pipet Tips
  - ddPCR 96 Well Plates
  - ddPCR Pierceable Foil Seals
- ddPCR Dx AutoDG Droplet Reader Oil Pack

Components of the kit QXDx BCR-ABL %IS KIT:

QXDX™ BCR-ABL primers & probes: Deoxyoligonucleotide primers and dye- and quencher- conjugated probes. Provides primers and probes for PCR amplification and detection of target sequences.
QXDX™ Nuclease Free Water: Nuclease Free Water. Adjust volume of RT & ddPCR reactions.
QXDX™ iScript Advanced Reverse Transcriptase: Reverse Transcriptase. Generate cDNA from RNA template.
QXDX™ 5x iScript Select Reaction Mix: Buffer for Reverse Transcriptase with salts, dNTPs, and glycerol. Reaction mix component of the RT reaction to generate cDNA from RNA template.
QXDX™ RT Primers: Reverse Transcriptase Primers. Random Primers used to prime the RT reaction to generate cDNA from RNA template.
QXDX™ 2X ddPCR™ Supermix: DNA polymerase, salt buffer, dNTPs, glycerol, and surfactants. Catalyzes the amplification of primers hybridized to templates from the cDNA. Enzyme exonuclease activity degrades hybridized probes to release fluorescence for detection of amplicons in each PCR cycle.
QXDX™ BCR-ABL ~0.1%IS: BCR-ABL and ABL RNA formulated to approximately 0.10% BCR-ABL/ABL. Per run controls to check against acceptance criteria for use of electronic WHO-IS CF factor and reporting of WHO-IS value results.
QXDX™ BCR-ABL ~10%IS: BCR-ABL and ABL RNA formulated to approximately 10% BCR-ABL/ABL.
QXDX™ BCR-ABL Neg-CTRL: ABL RNA. Control used to ensure that RT and PCR steps performed properly and protect against contamination and falsely positive samples due to contamination.
QXDX™ BCR-ABL H-CTRL: BCR-ABL and ABL RNA formulated to approximately 18% BCR-ABL/ABL. Control used to ensure that RT and ddPCR steps performed properly by generating expected MR value.
QXDX™ BCR-ABL L-CTRL: BCR-ABL and ABL RNA formulated to approximately .03% BCR-ABL/ABL.

Mentions image processing

Not Found

Mentions AI, DNN, or ML

Not Found

Input Imaging Modality

Not Found

Anatomical Site

Whole blood

Indicated Patient Age Range

Not Found

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

Not Found

Summary of Performance Studies

Precision and Multisite Reproducibility (CLSI EP15-A3)

Study Type: Precision and Reproducibility study.
Sample Size: Two panels of eight test samples each (six contrived samples and two controls). Contrived samples prepared by mixing RNA from BCR-ABL negative and positive whole blood specimens (e13a2 and e14a2 variants) in BCR-ABL:ABL ratios spanning the assay range. Control samples prepared from a BCR-ABL negative cell line and a BCR-ABL positive cell line (e13a2 variant). The reproducibility samples were prepared by Bio-Rad.
Data Source: Contrived and cell line samples prepared by Bio-Rad.
Key Results: 576 observations included in variance components analysis. Very low variability, within-site precision, and all acceptance criteria satisfied (all CVs 0.99) and slope close to 1.0.

Carryover Contamination

Study Type: Carryover Contamination.
Sample Size: 2, 96-well plates with high positive and negative wells in alternating rows. 4 rows each containing 12 replicates (48 carry over events) per plate. 2 plates tested on each of 3 QXDx™ DR instruments (total test sample size of 288).
Key Results: Of 288 replicate carryover tests, 286 met inclusion criteria. Only 1 negative well out of 285 showed signal (1 copy of BCR-ABL and 0 copies of ABL). No signal in remaining 285 negative wells. High occupancy rate (>99%) verified instrument operation.

Clinical Performance (Method Comparison)

Study Type: Clinical Evaluation Method Comparison.
Sample Size: 139 samples representing the intended use population, spanning the common dynamic range.
Data Source: De-identified leftover RNA samples previously collected from a minimum of two geographically distinct sites.
Key Results: Mean bias (95%CI) between Bio-Rad and Asuragen using Bland-Altman was 0.16 (0.14 to 0.19). Bio-Rad QXDx BCR-ABL %IS assay showed excellent correlation with the Comparator Kit (Asuragen Quantidex qPCR BCR-ABL IS Kit (IVD)) using weighted Deming regression with a Pearson R correlation coefficient of 0.99, slope 1.037, and intercept 0.1084.

Key Metrics

Precision and Reproducibility:

Total CV:

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

February 13, 2019

Image /page/0/Picture/1 description: The image contains the logo of the U.S. Food and Drug Administration (FDA). On the left is the Department of Health & Human Services logo. To the right of that is a blue square with the letters "FDA" in white. To the right of the blue square is the text "U.S. FOOD & DRUG ADMINISTRATION" in blue.

Bio-Rad Laboratories, Inc. Steve Lin Director of Regulatory Affairs and Quality Assurance 5731 W. Las Positas Blvd Pleasanton, California 94588

Re: K181661

Trade/Device Name: QXDx BCR-ABL %IS Kit for use on the QXDx AutoDG ddPCR System Regulation Number: 21 CFR 866.6060, 862.2570 Regulation Name: BCR-ABL quantitation test Regulatory Class: Class II Product Code: OYX, PHG Dated: January 3. 2019 Received: January 11, 2019

Dear Steve Lin:

We have reviewed your Section 510(k) premarket notification of intent to market the device referenced above and have determined the device is substantially equivalent (for the indications for use stated in the enclosure) to legally marketed predicate devices marketed in interstate commerce prior to May 28, 1976, the enactment date of the Medical Device Amendments, or to devices that have been reclassified in accordance with the provisions of the Federal Food, Drug, and Cosmetic Act (Act) that do not require approval of a premarket approval application (PMA). You may, therefore, market the device, subject to the general controls provisions of the Act. Although this letter refers to your product as a device, please be aware that some cleared products may instead be combination products. The 510(k) Premarket Notification Database located at https://www.accessdata.fda.gov/scripts/cdrh/cfdocs/cfpmn/pmn.cfm identifies combination product submissions. The general controls provisions of the Act include requirements for annual registration, listing of devices, good manufacturing practice, labeling, and prohibitions against misbranding and adulteration. Please note: CDRH does not evaluate information related to contract liability warranties. We remind you, however, that device labeling must be truthful and not misleading.

If your device is classified (see above) into either class II (Special Controls) or class III (PMA), it may be subject to additional controls. Existing major regulations affecting your device can be found in the Code of Federal Regulations, Title 21, Parts 800 to 898. In addition, FDA may publish further announcements concerning your device in the Federal Register.

Please be advised that FDA's issuance of a substantial equivalence determination does not mean that FDA has made a determination that your device complies with other requirements of the Act or any Federal statutes and regulations administered by other Federal agencies. You must comply with all the Act's requirements, including, but not limited to: registration and listing (21 CFR Part 807); labeling (21 CFR Part 801 and Part 809); medical device reporting of medical device-related adverse events) (21 CFR

1

for devices or postmarketing safety reporting (21 CFR 4, Subpart B) for combination products (see https://www.fda.gov/CombinationProducts/GuidanceRegulatoryInformation/ucm597488.htm); good manufacturing practice requirements as set forth in the quality systems (QS) regulation (21 CFR Part 820) for devices or current good manufacturing practices (21 CFR 4, Subpart A) for combination products; and, if applicable, the electronic product radiation control provisions (Sections 531-542 of the Act); 21 CFR 1000-1050.

Also, please note the regulation entitled, "Misbranding by reference to premarket notification" (21 CFR Part 807.97). For questions regarding the reporting of adverse events under the MDR regulation (21 CFR Part 803), please go to http://www.fda.gov/MedicalDevices/Safety/ReportaProblem/default.htm.

For comprehensive regulatory information about medical devices and radiation-emitting products, including information about labeling regulations, please see Device Advice (https://www.fda.gov/MedicalDevices/DeviceRegulationandGuidance/) and CDRH Learn (http://www.fda.gov/Training/CDRHLearn). Additionally, you may contact the Division of Industry and Consumer Education (DICE) to ask a question about a specific regulatory topic. See the DICE website (http://www.fda.gov/DICE) for more information or contact DICE by email (DICE@fda.hhs.gov) or phone (1-800-638-2041 or 301-796-7100).

Sincerely,

Yun-fu Hu -S

for Reena Philip, Ph.D. Director Division of Molecular Genetics and Pathology Office of In Vitro Diagnostics and Radiological Health Center for Devices and Radiological Health

Enclosure

2

Indications for Use

510(k) Number (if known)

K181661

Device Name

QXDx BCR-ABL %IS Kit for use on the QXDx AutoDG ddPCR System

Indications for Use (Describe)

The QXDx™ BCR-ABL %IS Kit is an in vitro nucleic acid amplification test for the quantitation of BCR-ABL and ABL1 transcripts in total RNA from whole blood of diagnosed (9;22) positive Chronic Myeloid Leukemia (CML) patients expressing BCR-ABL1 fusion transcripts type e13a2 and/or e14a2. The QXDx BCR-ABL %IS Kit is a reverse transcription-quantitative PCR performed on the Bio-Rad QXDx™ AutoDG™ ddPCR System 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 (9;22) positive CML patients during monitoring of treatment with Tyrosine Kinase Inhibitors (TKIs).

The QXDx BCR-ABL %IS Kit is intended for use only on the Bio-Rad QXDx AutoDG ddPCR System.

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.

Type of Use (Select one or both, as applicable)
Prescription Use (Part 21 CFR 801 Subpart D) Over-The-Counter Use (21 CFR 801 Subpart C)	Prescription Use (Part 21 CFR 801 Subpart D)	Over-The-Counter Use (21 CFR 801 Subpart C)
Prescription Use (Part 21 CFR 801 Subpart D)
Over-The-Counter Use (21 CFR 801 Subpart C)

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3

1. Submitter

Bio-Rad Laboratories, Inc. Establishment Registration Number: N/A 5731 W. Las Positas Blvd Pleasanton, CA 94566 Contact Name: Steve Lin Phone Number: (925) 474-9018 Fax Number: (925) 474-8644 Email: steve_lin@bio-rad.com Summary was prepared on February 10, 2019

2. Name of Device

| Trade name: | QXDx BCR-ABL %IS Kit for use on the QXDx
AutoDG ddPCR System |
|-------------------------|-----------------------------------------------------------------------------------------------------------------------------------------------------------|
| Common name: | BCR-ABL1 Digital PCR Test |
| Classification
Name: | Bcr/Abl1 Monitoring Test, OYX (per 21 CFR section
866.6060); Instrumentation For Clinical Multiplex
Test Systems, PHG (per 21 CFR section 862.2570) |

3. Predicate Devices

| Device Name | Premarket
Notification |
|-------------------------------------------------------------------------------|---------------------------|
| QuantideX qPCR BCR-ABL IS Kit | De Novo
DEN160003 |
| Applied Biosystems 7500 Fast Dx Real-Time PCR
Instrument with SDS Software | Class II K141220 |

4. Device Description

4

QXDx AutoDG ddPCR SYSTEM

The QXDx AutoDG ddPCR System consists of two instruments, the QXDx Automated Droplet Generator and the QXDx Droplet Reader, and their associated consumables. The QXDx Automated Droplet Generator partitions samples into approximately 20,000 nanoliter-sized droplets and, after PCR on a thermal cycler, droplets from each sample are analyzed individually on the QXDx Droplet Reader. PCR-positive and PCR-negative droplets are counted to provide direct quantification of nucleic acid in digital form. Results are analyzed on QXDx Software running on a Windows based computer.

The QXDx AutoDG ddPCR System contains:

QXDx Automated Droplet Generator ●
QXDx Droplet Reader ●
Laptop Computer with QXDx Software .
Accessory components: ●
- o ddPCR Dx AutoDG Consumable Pack
  - Automated Droplet Generation Oil for Probes ■
  - DG32 Cartridges w/ Gaskets ■
  - ddPCR Pipet Tips
  - . ddPCR 96 Well Plates
  - . ddPCR Pierceable Foil Seals
- o ddPCR Dx AutoDG Droplet Reader Oil Pack

QXDx BCR-ABL %IS KIT

Components of the kit QXDx BCR-ABL %IS KIT:

Item	Description	Use
QXDXTM BCR-ABL
primers & probes	Deoxyoligonucleotide
primers and dye- and
quencher- conjugated
probes.	Provides primers and
probes for PCR
amplification and
detection of target
sequences.
QXDXTM Nuclease Free
Water	Nuclease Free Water	Adjust volume of RT &
ddPCR reactions
QXDXTM iScript
Advanced Reverse
Transcriptase	Reverse Transcriptase	Generate cDNA from
RNA template
QXDXTM 5x iScript
Select Reaction Mix	Buffer for Reverse
Transcriptase with
salts, dNTPs, and
glycerol	Reaction mix
component of the RT
reaction to generate
cDNA from RNA
		template
QXDXTM RT Primers	Reverse Transcriptase
Primers	Random Primers used
to prime the RT
reaction to generate
cDNA from RNA
template
QXDXTM 2X ddPCRTM
Supermix	DNA polymerase, salt
buffer, dNTPs, glycerol,
and surfactants	Catalyzes the
amplification of primers
hybridized to templates
from the cDNA.
Enzyme exonuclease
activity degrades
hybridized probes to
release fluorescence
for detection of
amplicons in each PCR
cycle.
QXDXTM BCR-ABL
~0.1%IS	BCR-ABL and ABL
RNA formulated to
approximately 0.10%
BCR-ABL/ABL	Per run controls to
check against
acceptance criteria for
use of electronic WHO-
IS CF factor and
reporting of WHO-IS
value results
QXDXTM BCR-ABL
~10%IS	BCR-ABL and ABL
RNA formulated to
approximately 10%
BCR-ABL/ABL
QXDXTM BCR-ABL
Neg-CTRL	ABL RNA	Control used to ensure
that RT and PCR steps
performed properly and
protect against
contamination and
falsely positive samples
due to contamination
QXDXTM BCR-ABL H-
CTRL	BCR-ABL and ABL
RNA formulated to
approximately 18%
BCR-ABL/ABL	Control used to ensure
that RT and ddPCR
steps performed
properly by generating
expected MR value
QXDXTM BCR-ABL L-
CTRL	BCR-ABL and ABL
RNA formulated to
approximately .03%
BCR-ABL/ABL

5

5. Indications for Use

6

The QXDx™ 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 QXDx BCR-ABL %IS Kit is a reverse transcription-quantitative PCR performed on the Bio-Rad QXDx™ AutoDG™ ddPCR System 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 QXDx BCR-ABL %IS Kit is intended for use only on the Bio-Rad QXDx AutoDG ddPCR System.

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.

6. Intended Use

Same as indications for use.

7. Substantial Equivalence Information:

The QXDx BCR-ABL %IS Kit for use on the QXDx AutoDG ddPCR System is substantially equivalent to the following legally marketed devices:

Device Name	Premarket Notification	Features	QXDx BCR-ABL %IS Kit
QuantideX qPCR BCR-ABL IS Kit	De Novo
DEN160003	Intended Use	Same	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.
Applied Biosystems 7500 Fast Dx Real-Time PCR
Instrument with SDS Software	Class II K141220

The table on the following pages compares the technical characteristics of the QXDx BCR-ABL %IS Kit for use on the QXDx AutoDG ddPCR System. As can be seen from the table, the QXDx BCR-ABL %IS Kit for use on the QXDx AutoDG ddPCR System is substantially equivalent in technological characteristics.

7

Comparison Table of Predicate and Subject Device

8

| Platform | QXDx AutoDG ddPCR
System | Applied Biosystems 7500
Fast Dx Real-Time PCR
Instrument with SDS
Software |
|---------------------------------------------------------------------|-----------------------------------------------------------------------------------------------------------|----------------------------------------------------------------------------------------------------------------------------------------------------------------------------|
| Measuring Range | Same | MR 0.3 to MR 4.7 |
| Specimen Type | Same | Human whole blood |
| Assay Principle | Droplet Digital PCR | Real-Time PCR |
| Matrices | Same | EDTA |
| Standardization | Same | WHO-IS |
| | | |
| Features | QXDx AutoDG ddPCR
System | Applied Biosystems 7500
Fast Dx Real-Time PCR
Instrument with SDS
Software |
| Fundamental
Technology | Digital PCR | Real-Time PCR |
| Multiplex capable | Same | Able to measure and sort
multiple signals
generated by an assay
from a clinical sample. |
| | | |
| Instrument Computer
Operating System | Microsoft Windows 10 | Microsoft Windows 7 |
| Degree of Automation | Same, except amplification
functionality is not included | Requires manual transfer of
amplification mixture to
amplification/detection
instrument.
Automated control of
amplification, detection and
data analysis |
| Primary Operational
Amplification and
Detection
Components | Amplification functionality is
not included.
Nanoliter droplet fluorimeter
for walk away PCR
| Integrated thermal cycler
and microvolume
fluorimeter for walk
away PCR amplification
and detection |
| Amplification Reaction
Volume | 20-25 uL in 96-well Bio-Rad
PCR plates | 10-30 uL in 96-well Fast
PCR plates |

9

Conclusion:

Differences in technological characteristics do not raise any questions of safety and effectiveness. The subject device is substantially equivalent to the predicate device.

8. Principles of the Procedure

Testing begins with RNA purified from peripheral whole blood samples (see Section 9 for proposed sample preparation method). The RNA sample and iScript reverse transcription reagents are combined to produce complementary DNA (cDNA), which is then added to the ddPCR Supermix to prepare the PCRready sample.

25 microliters of the PCR-ready sample is loaded into a 96-well PCR plate. The plate, as well as required consumables (Automated Droplet Generation Oil for Probes, DG32 Cartridges w/ Gaskets and ddPCR Pipet Tips) are loaded into the QXDx Automated Droplet Generator. The QXDx Automated Droplet Generator

10

uses microfluidics to combine oil and aqueous sample to generate the nanolitersized droplets required for ddPCR analysis.

The 96-well PCR plate containing droplets from the QXDx Automated Droplet Generator is sealed with foil and a plate sealer, and thermal cycled to end point (~40cycles) using a thermal cycler.

The thermal cycled plate is loaded into the QXDx Droplet Reader. The Droplet Reader singulates the droplets and streams them in single file past a two-color detector. The detector reads the droplets to determine which contain target (positive) and which do not (negative).

The QXDx Droplet Reader connects to a laptop computer running QXDx Software. The software provides measured levels of BCR-ABL and reference gene (BCR-ABL/ABL concentration ratio), quality values, World Health Organization International Scale (IS) calibrated results and quality associated with any controls or calibrators run.

9. 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 normallydistributed data.

Another value commonly reported in the literature is the Molecular Reduction, or MR value. The MR value is traditionally written as MR**. However, for simplicity and legibility, the OXDx BCR-ABL %IS Kit will report the value as MRx.x. The MR value is the log10 reduction from the internationally standardized baseline, defined as 100% IS. Therefore,

MRx.x = log10(100/%IS) = log10(100) - log10(%IS) = 2 - log10(%IS)

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 show below:

MR	%IS
0.0	100

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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

10. Analytical Performance

Precision and Multisite Reproducibility (CLSI EP15-A3) – instrument to instrument and operator to operator reproducibility

Precision and Reproducibility was assessed using two panels of eight test samples each. Each panel consisted of six contrived samples and two controls. The contrived samples were prepared by mixing six independent pools of RNA isolated from BCR-ABL negative whole blood specimens with six independent pools of RNA isolated from BCR-ABL positive whole blood specimens, representing both e13a2 and e14a2 variants, in BCR-ABL:ABL ratios that spanned the assay range. The control samples were prepared by mixing RNA isolated from a BCR-ABL negative cell line and RNA isolated from a BCR-ABL positive cell line (e13a2 variant). The reproducibility samples were prepared by Bio-Rad.

Samples were assayed in 2 replicates per run for 2 runs per day for 3 nonconsecutive days at 3 sites (one instrument at each site) with one reagent lot (2 reps x 2 runs x 3 days x 3 sites/instruments x 1 reagent lot = 36 replicates). Each run was performed by an independent operator (2 operators per site).

Acceptance Criteria:

When samples are 1ug +/- 25% RNA yielding >100,000 ABL copies and loads ranging from 640ng/test

Total CV must be o 99%. Of the two-hundred and eighty-six (286) replicates used in the analysis, signal was measured in only one (1) negative well. One well contained 1 copy of BCR-ABL and 0 copies of ABL. No signal was measured in the remaining 285 negative wells.

11. Clinical Performance (Method Comparison)

A clinical evaluation method comparison study was designed to evaluate the performance of the QXDx BCR-ABL %IS Assay compared to the Asuragen Quantidex qPCR BCR-ABL IS Kit (IVD) in RNA derived from human blood in accordance with this protocol.

139 samples representing the intended use population and spanning the common dynamic range of the two methods were evaluated at a single testing lab.

Samples were acquired from at least two geographically distinct regions. The samples were collected and stored by the sites using a pre-specified protocol with eligibility criteria that fit this test including a system compatible RNA extraction method.

The participating site tested de-identified leftover RNA samples that have been previously collected from a minimum of two (2) sites. The samples were extracted RNA from peripheral whole blood. Sample testing occurred at one clinical site.

The mean bias (95%CI) between Bio-Rad and Asuragen using a Bland-Altman was 0.16 (0.14 to 0.19) indicating that the limits of agreement (LOA)

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between the two methods should lie between 0.14 and 0.19 for 95% of the time.

The Bio-Rad QXDx BCR-ABL %IS assay showed excellent correlation with the Comparator Kit using a weighted Deming regression with a Pearson R correlation coefficient of 0.99, slope 1.037 and intercept was 0.1084.

12. Conclusion from the Clinical and Nonclinical Tests

The device is as safe, as effective, and performs as well as or better than the predicate device. The device is substantially equivalent to the predicate device.