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 (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.

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: ●
- 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

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

QXDXTM BCR-ABL primers & probes
QXDXTM Nuclease Free Water
QXDXTM iScript Advanced Reverse Transcriptase
QXDXTM 5x iScript Select Reaction Mix
QXDXTM RT Primers
QXDXTM 2X ddPCRTM Supermix
QXDXTM BCR-ABL ~0.1%IS
QXDXTM BCR-ABL ~10%IS
QXDXTM BCR-ABL Neg-CTRL
QXDXTM BCR-ABL H-CTRL
QXDXTM BCR-ABL L-CTRL

AI/ML Overview

The provided document details the analytical and clinical performance of the QXDx BCR-ABL %IS Kit for use on the QXDx AutoDG ddPCR System. This device 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 Chronic Myeloid Leukemia (CML) patients.

The document does not describe the acceptance criteria and study for an AI/ML powered medical device, but rather for an in vitro diagnostic (IVD) kit. Therefore, many of the typical acceptance criteria and study elements associated with AI/ML devices (e.g., human-in-the-loop performance, expert consensus for ground truth on images, MRMC studies) are not applicable.

Below is a breakdown of the acceptance criteria and study details provided for this IVD kit, adapted to the requested format where possible, and noting where specific requested information (relevant to AI/ML devices) is not available for this type of device.

Acceptance Criteria and Reported Device Performance

The core acceptance criteria are related to the analytical performance of the kit, primarily precision, reproducibility, cross-reactivity, interference, linearity, and detection capability. The performance is consistently reported in terms of Molecular Reduction (MR) value or %International Scale (%IS), which are standardized measures for BCR-ABL levels.

Table 1: Acceptance Criteria of the QXDx BCR-ABL %IS Kit and Reported Performance

Category / Study	Acceptance Criteria	Reported Device Performance
1. Precision & Reproducibility	Total CV (Reproducibility):	Total CV (Reproducibility):
	- <50% at LoQ	- Max %CV for LoQ samples (S12) was 4.84% (Success)
	- <10% at MR 0.3 – 2.49	- Max %CV for MR 0.3-2.49 samples (S07) was 4.52% (Success)
	- ≤20% at MR 2.5 – 3.49	- Max %CV for MR 2.5-3.49 samples (S05) was 2.57% (Success)
	- <50% at MR 3.5 – 4	- Max %CV for MR 3.5-4 samples (S12) was 4.84% (Success)
	Within Run CV (Repeatability): <15% at MR 3	Within Run CV (Repeatability): All CVs were less than 5.0% (Success)
	Between Instrument (site) CV: <15% at MR 3	Between Instrument (site) CV: All CVs were less than 5.0% (Success)
	Between Day CV (Within-site Precision): <15% at MR 3	Between Day CV (Within-site Precision): All CVs were less than 5.0% (Success)
	Between Operator (run) CV (Within day precision): <15% at MR 3	Between Operator (run) CV (Within day precision): All CVs were less than 5.0% (Success)
2. Lot to Lot Reproducibility	All samples (patient, cell line, in-kit calibrators/controls) met specified CVs (not explicitly listed here, but implied by text, except for issues with very low %IS/MR samples preventing reliable calculation).	All included samples met acceptance criteria. (Max CV 15% for MR3 samples)
3. Cross Reactivity	QXDx™ BCR-ABL %IS measured in non-target variants (p190, p230) should be 0.000%. (Implied: expected result for cross-reactivity is absence of signal.)	QXDx™ BCR-ABL %IS measured in all p190 and p230 variant samples was 0.000%. (PASS for all dilutions, 100% Specificity reported).
4. Interference	For MR values: Mean test MR value must fall within 95% confidence interval plus or minus 0.5 log of the control. For %IS: 95% confidence interval of the mean test %IS must intersect the within-run precision range for control samples.	All cases for both MR and %IS passed the acceptance criteria. For %IS, not only did the test 95% CI intersect the control precision range, but the test mean %IS fell within the within-run precision range.
5. Assay Linearity	Slope (m): 0.8 - 1.2 R2: 0.97 - 1.0 Range %IS: 10% - 0.1% (for e13a2 and e14a2 variants)	E13a2: m=1.04, R2=0.996, Range = 50%-0.002% (PASS) E14a2: m=1.01, R2=0.992, Range = 50%-0.002% (PASS) (The stated range for test appears broader than acceptance criteria but still within acceptable limits, potentially due to the nature of the samples created for the study)
6. Detection Capability (LoD)	If percentage of tests results at or below the LoB was ≤ 5%, then the LoD was the concentration of the test sample (least MR value).	LoD was determined to be MR 4.7 (0.002% IS BCR-ABL). For e13a2, 98.1% tests were above LoB (median MR 4.7). For e14a2, 99.4% tests were above LoB (median MR 4.7). (PASS).
7. Kit, Calibrator & Control Stability	No specific numerical acceptance criteria shown here, but stated that kits must meet acceptance criteria at specified time points. (Implied: results should remain within expected ranges/precision).	Real-time stability: Met acceptance criteria at 12 months for Lot D, and 5 months for Lots H and I (ongoing study). Freeze-thaw stability: Stable performance for at least 5 freeze-thaw cycles.
8. Specimen Stability(Whole Blood)	Allowable Range: Mean MR value at Day 1 ± 0.5 log. Pass if the 95% CI for each test sample fell entirely within the allowable range.	All samples tested met the specification.
9. WHO Standard Quantification	Slope (m): 0.95-1.05 Correlation (r2): 0.98-1.00 Intercept (b): -0.2-0.2 (Compared against WHO assigned values via regression analyses).	All 7 kit lots tested met the acceptance criteria for slope, correlation, and intercept. Overall measured values (m=1.014, r2=0.995, b=-0.006) indicate strong alignment.
10. Carryover Contamination	Implied: Minimal to no signal in negative wells when alternating with high positive wells.	Of 286 valid replicates, signal was measured in only one (1) negative well (1 copy of BCR-ABL, 0 ABL). The remaining 285 negative wells had no signal. This demonstrates minimal carryover.
11. Clinical Performance (Method Comparison)	Demonstrates substantial equivalence to predicate device. No specific numerical criteria for Bland-Altman or Deming regression provided in the listed acceptance criteria section, but study results are reported.	Bland-Altman: Mean bias (95%CI) between Bio-Rad and Asuragen was 0.16 (0.14 to 0.19) MR. Deming regression: Pearson R correlation coefficient of 0.99, slope 1.037, intercept 0.1084. (Indicates excellent correlation).

Study Details for the QXDx BCR-ABL %IS Kit

As this is an in vitro diagnostic (IVD) kit and not an AI/ML powered device, several of the requested sections are not applicable.

Sample sizes used for the test set and data provenance:
- Precision and Multisite Reproducibility: 36 replicates per sample (2 reps x 2 runs x 3 days x 3 sites x 1 reagent lot). Two panels of eight test samples each (six contrived, two controls).
- Lot to Lot Reproducibility: 108 data points per sample (3 replicates x 2 operators x 3 days x 3 lots x 2 instruments). Sixteen (16) BCR-ABL1 negative and one hundred (100) BCR-ABL1 positive RNA samples (pooled).
- Cross Reactivity: Each dilution tested with 4 or 8 replicates (N). Two prepared samples (p190, p230 variants).
- Interference: Ten (10) tests per sample type (5 replicate extractions x 2 tests per extraction). Various interferents tested.
- Assay Linearity: Not explicitly stated, but typically involves multiple dilutions. Two positive BCR-ABL RNA patient pools (E13a2, E14a2) and one negative RNA pool.
- Detection Capability (LoD): 160 replicates per sample (20 replicates x 4 days x 2 lots x 1 instrument) for two contrived samples (e13a2, e14a2 variants).
- Kit, Calibrator and Control Stability: 3 kit lots at various time points (T0, T1, T2.5, T5, T11, T12, T19, T25 months). Freeze-thaw: 4 kits from one lot tested through several cycles.
- Specimen Stability (Whole Blood Stability): Positive blood sample and 3 negative blood samples. Dilutions tested with 4 or 11 replicate extractions.
- WHO Standard Quantification: 7 kit lots. Four (4) levels of WHO primary standards tested in four (4) replicates per lot.
- Carryover Contamination: 288 initial replicate tests; 286 valid replicates (from 2 plates, tested on 3 instruments).
- Clinical Performance (Method Comparison): 139 samples. Samples acquired from at least two geographically distinct regions. Samples were de-identified leftover RNA samples, previously collected. The study was conducted at a single testing lab.
- Data Provenance (Retrospective/Prospective, Country): For the clinical method comparison study, samples were "de-identified leftover RNA samples that have been previously collected from a minimum of two (2) sites." This indicates a retrospective data collection approach. The country of origin is not explicitly stated, but the submission is to the US FDA, so it's likely from the US or a region with compliant medical standards.
Number of experts used to establish the ground truth for the test set and the qualifications of those experts:
- Not Applicable. For this IVD device, the "ground truth" is established by the known concentrations/compositions of the samples (e.g., contrived samples with specific BCR-ABL:ABL ratios, WHO primary reference standards with assigned values, or comparison to a legally marketed predicate device). There are no human experts "reading" or annotating images or clinical data to establish ground truth in the way described for AI/ML devices.
Adjudication method (e.g. 2+1, 3+1, none) for the test set:
- Not Applicable. As there are no human expert readings of the test set requiring adjudication. The ground truth refers to the biochemical composition or an independent, established reference assay.
If a multi-reader multi-case (MRMC) comparative effectiveness study was done, If so, what was the effect size of how much human readers improve with AI vs without AI assistance:
- Not Applicable. This is an in vitro diagnostic device, not an AI-powered system designed to assist human readers (e.g., radiologists interpreting images). The "method comparison" study compares the device's analytical performance against a predicate device, not human performance.
If a standalone (i.e. algorithm only without human-in-the-loop performance) was done:
- This entire document describes the standalone performance of the IVD kit and its associated system (QXDx AutoDG ddPCR System). The output is a quantitative MR value or %IS, which is then used by clinicians for patient management. There is no "human-in-the-loop performance" for the device's operation itself, beyond standard laboratory procedures and interpretation of the quantitative results.
The type of ground truth used (expert consensus, pathology, outcomes data, etc.):
- The ground truth extensively used in the analytical studies is based on:
  - Contrived samples: Samples prepared by mixing known concentrations of BCR-ABL positive and negative RNA, allowing for precise control of target ratios.
  - Reference materials: Specific WHO International Standards (WHO-IS primaries) with assigned values used for calibration and verification.
  - Predicate device comparison: The Asuragen Quantidex qPCR BCR-ABL IS Kit (IVD) served as the comparator in the clinical method comparison study, establishing a "ground truth" for comparative performance.
  - Known sample characteristics: E.g., for cross-reactivity and interference studies, samples with known absence of target or presence of interfering substances are used.
The sample size for the training set:
- Not applicable in the usual AI/ML sense. This is an IVD kit involving biochemical reactions and a detection system, not a machine learning algorithm that undergoes 'training' on a dataset in the same way. The development and optimization of the kit's components and parameters would be based on internal R&D, but there isn't a "training set" in the context of typical AI/ML submissions.
How the ground truth for the training set was established:
- Not Applicable. As above, there's no "training set" in the AI/ML sense. The ground truth for the analytical studies (which could be considered analogous to validation/testing stages for IVDs) was established by carefully prepared samples with known concentrations/compositions or recognized international standards.

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.

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)

CONTINUE ON A SEPARATE PAGE IF NEEDED.

This section applies only to requirements of the Paperwork Reduction Act of 1995.

DO NOT SEND YOUR COMPLETED FORM TO THE PRA STAFF EMAIL ADDRESS BELOW.

The burden time for this collection of information is estimated to average 79 hours per response, including the time to review instructions, search existing data sources, gather and maintain the data needed and complete and review the collection of information. Send comments regarding this burden estimate or any other aspect of this information collection, including suggestions for reducing this burden, to:

Department of Health and Human Services Food and Drug Administration Office of Chief Information Officer Paperwork Reduction Act (PRA) Staff PRAStaff(@fda.hhs.gov

"An agency may not conduct or sponsor, and a person is not required to respond to, a collection of information unless it displays a currently valid OMB number."

{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 QXDxAutoDG ddPCR System
Common name:	BCR-ABL1 Digital PCR Test
ClassificationName:	Bcr/Abl1 Monitoring Test, OYX (per 21 CFR section866.6060); Instrumentation For Clinical MultiplexTest Systems, PHG (per 21 CFR section 862.2570)

3. Predicate Devices

Device Name	PremarketNotification
QuantideX qPCR BCR-ABL IS Kit	De NovoDEN160003
Applied Biosystems 7500 Fast Dx Real-Time PCRInstrument with SDS Software	Class II K141220

4. Device Description

{4}------------------------------------------------

QXDx AutoDG ddPCR SYSTEM

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-ABLprimers & probes	Deoxyoligonucleotideprimers and dye- andquencher- conjugatedprobes.	Provides primers andprobes for PCRamplification anddetection of targetsequences.
QXDXTM Nuclease FreeWater	Nuclease Free Water	Adjust volume of RT &ddPCR reactions
QXDXTM iScriptAdvanced ReverseTranscriptase	Reverse Transcriptase	Generate cDNA fromRNA template
QXDXTM 5x iScriptSelect Reaction Mix	Buffer for ReverseTranscriptase withsalts, dNTPs, andglycerol	Reaction mixcomponent of the RTreaction to generatecDNA from RNA
		template
QXDXTM RT Primers	Reverse TranscriptasePrimers	Random Primers usedto prime the RTreaction to generatecDNA from RNAtemplate
QXDXTM 2X ddPCRTMSupermix	DNA polymerase, saltbuffer, dNTPs, glycerol,and surfactants	Catalyzes theamplification of primershybridized to templatesfrom the cDNA.Enzyme exonucleaseactivity degradeshybridized probes torelease fluorescencefor detection ofamplicons in each PCRcycle.
QXDXTM BCR-ABL~0.1%IS	BCR-ABL and ABLRNA formulated toapproximately 0.10%BCR-ABL/ABL	Per run controls tocheck againstacceptance criteria foruse of electronic WHO-IS CF factor andreporting of WHO-ISvalue results
QXDXTM BCR-ABL~10%IS	BCR-ABL and ABLRNA formulated toapproximately 10%BCR-ABL/ABL
QXDXTM BCR-ABLNeg-CTRL	ABL RNA	Control used to ensurethat RT and PCR stepsperformed properly andprotect againstcontamination andfalsely positive samplesdue to contamination
QXDXTM BCR-ABL H-CTRL	BCR-ABL and ABLRNA formulated toapproximately 18%BCR-ABL/ABL	Control used to ensurethat RT and ddPCRsteps performedproperly by generatingexpected MR value
QXDXTM BCR-ABL L-CTRL	BCR-ABL and ABLRNA formulated toapproximately .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.

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
QuantideX qPCR BCR-ABL IS Kit	De NovoDEN160003	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 PCRInstrument 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 ddPCRSystem	Applied Biosystems 7500Fast Dx Real-Time PCRInstrument with SDSSoftware
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 ddPCRSystem	Applied Biosystems 7500Fast Dx Real-Time PCRInstrument with SDSSoftware
FundamentalTechnology	Digital PCR	Real-Time PCR
Multiplex capable	Same	Able to measure and sortmultiple signalsgenerated by an assayfrom a clinical sample.

Instrument ComputerOperating System	Microsoft Windows 10	Microsoft Windows 7
Degree of Automation	Same, except amplificationfunctionality is not included	Requires manual transfer ofamplification mixture toamplification/detectioninstrument.Automated control ofamplification, detection anddata analysis
Primary OperationalAmplification andDetectionComponents	Amplification functionality isnot included.Nanoliter droplet fluorimeterfor walk away PCR	Integrated thermal cyclerand microvolumefluorimeter for walkaway PCR amplificationand detection
Amplification ReactionVolume	20-25 uL in 96-well Bio-RadPCR plates	10-30 uL in 96-well FastPCR 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

{11}------------------------------------------------

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 <50% at LoQ

{12}------------------------------------------------

o MR0.3-2.49 < 10%
o MR2.5-3.49 ≤ 20%
o MR3.5-4 < 50%
Within run (CV <15% at MR3) ●
Between instrument (CV<15% at MR3) ●
Between day (CV <15% at MR3) ●
Between operator (CV <15% at MR3) ●

A total of 576 observations were included in a variance components analysis with random effects for site, day, and run to assess repeatability, within-day precision, within-site precision, and reproducibility of measured MR level. Results shown on the following table indicated very low variability, including within sites, and all acceptance criteria were satisfied (all CVs were < 15%).

Criteria	Conclusion
-Total CV(Reproducibility)	Success(S012 %CV was 4.84)
≤ 50% at LoQ
≤ 10% at MR 0.3 –2.49	Success(maximum %CV was for S07 at4.52)
≤ 20% at MR 2.5 –3.49	Success(maximum %CV was for S05 at2.57)
≤ 50% at MR 3.5 – 4	Success(maximum %CV was for S12 at4.84)
Within run CV(Repeatability)-	Success – all CVs were less than5.0%
< 15% at MR 3
Between instrument(site) CV /(Reproducibility)	Success – all CVs were less than5.0%
< 15% at MR 3
Between day CV(Within-sitePrecision)-	Success – all CVs were less than5.0%
< 15% at MR 3
Between operator(run) CV(Within dayprecision)	Success - all CVs were less than5.0%
< 15% at MR 3

{13}------------------------------------------------

Lot to Lot Reproducibility (CLSI EP15-A3)

Sixteen (16) BCR-ABL1 negative whole blood and one-hundred (100) BCR-ABL1 positive RNA samples representing both the e13a2 and e14a2 variants were procured from commercial vendors. RNA was extracted from the whole blood samples and pooled to create six (6) negative patient RNA pools. Each negative pool used four (4) negative whole blood samples and each pool used at least 2 unique samples as compared to other pools. The positive RNA samples were pooled to create six (6) positive patient RNA pools. Each positive pool used minimally fifteen (15) samples each of which was unique to one (1) pool. The positive and negative RNA pools described above were blended with poly A RNA for stability. A high positive control sample (IHC 17%) was prepared by spiking Meg-01 (ATCC) BCR-ABL positive e13 variant cell line into a BCR-ABL negative HeLa cell line. A low positive control sample (MR4-5) was prepared by spiking BCR-ABL positive e14 variant cell line into a negative patient RNA pool and a BCR-ABL negative HeLa cell line.

Assay kit calibration checks and controls were tested in replicates of one (1) on each plate in order to qualify the run. Each sample, including control samples, was tested by 2 operators on 2 instruments and with 3 lots of reagents. All samples were tested in triplicate for 3 non-consecutive days. "Day 1" did not necessarily mean the same day. The mean, standard deviation (SD) and coefficient of variation (%CV) were calculated for each sample according to CLSI EP5-A2 prescribed methods for data analysis (3 replicates x 2 operators x 3 days x 3 lots x 2 instruments = 108 data points per sample).

Total MR and %IS precision was calculated for patient samples, cell line control samples and in-kit calibrators/controls. While the specifications as outlined were described for only MR, we evaluated total precision results for both measurement types across all samples. Cell line control 2 (MR4-5) led to an extremely low %IS (below LoQ) preventing a reliable calculation for %CV. thus, the data was not used. Similarly the in kit negative control also yielded a value too low for a realistic evaluation and was marked as "N/A – too low". All samples included in the evaluation met the acceptance criteria.

Additional precision metrics for both MR and %IS, including within-run, between run, between operator, between lot and between instruments are displayed for patient and cell line control samples only. As in-kit calibrator checks and controls were run in replicates of one (1) per plate, these metrics could not be calculated for these samples and total precision must represent overall performance. While the specification described a 15% or better CV across all metrics for samples close to MR3.0, we included all MR sample data in the evaluation. All samples met the specification as written for MR.

{14}------------------------------------------------

Cross Reactivity

Two (2) samples were prepared by blending in-vitro transcribed p190 or p230 with RNA extracted from normal, healthy, human blood from 2 donors. Four (4) dilutions of each sample were prepared by varying the amount of negative RNA used.

The QXDx™ BCR-ABL %IS measured in all samples was 0.000% demonstrating the kit detects neither the minor e1a2 (p190) nor micro e19a2 (p230) variants. All calibrators and controls met acceptance criteria.

VariantSampleID	DilutionNumber	N	MeanSpecific AssayRatio %	% CVSpecificAssay Ratio	MeanQXDx BCR-ABL%IS	% CVQXDx BCR-ABL %IS	%Specificity	Pass/Fail
p190	1	4	36.1329%	2.1%	0.000%	0.000%	100.000%	PASS
	2	4	0.7148%	5.3%	0.000%	0.000%	100.000%	PASS
	3	4	0.0749%	10.7%	0.000%	0.000%	100.000%	PASS
	4	8	0.0080%	42.8%	0.000%	0.000%	100.000%	PASS
p230	1	4	31.7429%	1.1%	0.000%	0.000%	100.000%	PASS
	2	4	0.6207%	2.5%	0.000%	0.000%	100.000%	PASS
	3	4	0.0630%	11.0%	0.000%	0.000%	100.000%	PASS
	4	8	0.0056%	63.4%	0.000%	0.000%	100.000%	PASS

Interference (CLSI EP07-A2)

For each test, a sample pool was prepared by mixing whole blood from a CML positive and CML negative patient. Samples were mixed on ice and stored less than 1 hour prior to extraction to prevent blood type incompatability hemolysis. For each interfering substance tested, the sample pool used was split into two (2) parts, a test and control. The potential interfering substance was added to the test pool and diluent was added to the control pool in concentrations recommended by CLSI EP7-A2.

Interferent	PositivePatientSample ID	Base %BCR-ABL/ABLResult	NegativePatientSample ID	ConcentrationTested	ControlDiluent Used	ExtractionDate
Cholesterol	CMLBR017	0.16%	M5366	6.47 mmol/L	3.8mmol/Lmethyl-ß-cyclodextrin	1/24/17
Conjugated Bilirubin	CMLBR017	0.16%	M5366	86 μM	PBS	1/24/17

{15}------------------------------------------------

Interferent	PositivePatientSample ID	Base %BCR-ABL/ABLResult	NegativePatientSample ID	ConcentrationTested	ControlDiluent Used	ExtractionDate
EDTA	CMLBR017	0.16%	M5366	7 mg/ml	PBS + 0.1NNaOH	1/25/17
Hemoglobin	CMLBR020	1.30%	85962	200 g/L	PBS	2/14/17
Sodium Heparin	CMLBR017	0.16%	M5366	3000 U/L	PBS	1/25/17
Triglycerides	CMLBR017	0.16%	M5366	5.6 mmol/L	2.5%glycerol innuclease-free water	1/24/17
Unconjugated Bilirubin	CMLBR017	0.16%	M5366	257μM	PBS + 0.1NNaOH	1/24/17

For both the control and test samples, five (5) replicate extractions were performed using the Promega Maxwell CSC RNA Blood Kit. Following extraction, absorbance at 260nm was measured and replicates normalized to 100ng/uL by adding DNA suspension buffer as needed. Each extracted sample was tested in replicates of two (2) for a total of ten (10) tests per sample type.

All in-kit calibrator check and control results met the run acceptance criteria. All samples were under MR3. For the MR values, the mean test MR value needed to fall within the 95% confidence interval plus or minus 0.5 log. In all cases, samples passed the acceptance criteria.

For the %IS data, the 95% confidence interval of the mean %IS for test samples needed to intersect the within run precision range for control samples. In all cases, not only did the test 95% confidence interval intersect the control precision range, but also the test mean %IS fell within the within run precision range, passing the acceptance criteria.

Test	Measured MeanMR		MR 95%Confidence Interval		MR AcceptableRange(Control CI ± 0.5Log)	Result (Does the TestSample MR CI fallwithin the ControlSample CI ± 0.5Log?)
	Control	Test	Control	Test
Cholesterol	4.38	4.38	4.04 - Inf*	3.97 - Inf*	3.54 - Inf*	PASS
Conjugated	4.30	4.15	4.00 - Inf*	3.84 - Inf*	3.50 - Inf*	PASS

MR test results, acceptance criteria and status.

{16}------------------------------------------------

Bilirubin
EDTA	3.00	3.03	2.85 - 3.25	2.83 - 3.38	2.35 - 3.75	PASS
Hemoglobin	3.34	3.35	3.21 - 3.54	3.26 - 3.46	2.71 - 4.04	PASS
Heparin	3.04	3.00	2.85 - 3.38	2.88 - 3.17	2.35 - 3.88	PASS
Triglycerides	4.21	4.41	3.89 - Inf*	4.02 - Inf*	3.39 - Inf*	PASS
UnconjugatedBilirubin	4.29	4.32	3.92 - Inf*	3.95 - Inf*	3.42 - Inf*	PASS

%IS test results, acceptance criteria and status.

Test	N		Mean %IS		%IS Control Precision %CV	%IS Control Precision Range	Does the Test %IS fall in the Control Precision Range?	Test %IS 95% Confidence interval	Does the Test %IS 95% CI intersect the Control Precision Range?
	Control	Test	Control	Test
Cholesterol	10	10	0.0042%	0.0042%	60.88%	0.0016% - 0.0067%	Yes	0.0000% - 0.0108%	Yes - Pass
ConjugatedBilirubin	10	10	0.0050%	0.0071%	51.42%	0.0024% - 0.0075%	Yes	0.0000% - 0.0144%	Yes - Pass
EDTA	10	10	0.0989%	0.0943%	22.20%	0.0769% - 0.1208%	Yes	0.0420% - 0.1466%	Yes - Pass
Hemoglobin	10	9*	0.0453%	0.0447%	18.24%	0.0370% - 0.0536%	Yes	0.0349% - 0.0546%	Yes - Pass
Heparin	10	10	0.0912%	0.1002%	27.43%	0.0662% - 0.1162%	Yes	0.0683% - 0.1321%	Yes - Pass
Triglycerides	10	10	0.0061%	0.0039%	56.99%	0.0026% - 0.0096%	Yes	0.0000% - 0.0095%	Yes - Pass
UnconjugatedBilirubin	10	10	0.0052%	0.0048%	67.75%	0.0017% - 0.0087%	Yes	0.0000% - 0.0113%	Yes - Pass

*One failing sample due to operator error

Assay Linearity (CLSI EP06-A)

Two (2) positive BCR-ABL RNA patient pools were prepared by mixing RNA extracted from BCR-ABL positive whole blood. Pool 1 contained RNA from five (5) patients positive for the E13a2 variant and Pool 2 contained RNA from five (5) patients positive for the E14a2 variant. The RNA concentration of each pool was measured by absorbance at 260nm and was brought to a final concentration of 100ng/μL with 3T3 mouse BCR-ABL1 negative RNA. One (1) negative BCR-ABL RNA sample pool was prepared by mixing RNA from fifteen (15) BCR-ABL negative whole blood samples with RNA extracted from a HeLa cell line to bring the final concentration to 100ng/μL. The negative RNA pool was used in the dilution of the positive patient sample pools.

Results from Linearity Study:

{17}------------------------------------------------

Variant	Regressionlineslope (m)	AcceptableRange, m	Result	R2	AcceptableRange, R2	Result	Range%IS	AcceptableRange, %IS	Result
E13a2	1.04	0.8-1.2	PASS	0.996	0.97-1.0	PASS	50%-0.002%	10%-0.1%	PASS
E14a2	1.01		PASS	0.992		PASS	50%-0.002%	10%-0.1%	PASS

Evaluation of Detection Capability (CLSI EP17-A2)

Two (2) contrived positive BCR-ABL RNA samples were prepared to achieve MR values at approximately MR4.7, as determined a priori in LoD by Probit. Pool 1 contained five (5) patients positive for the e13a2 variant and Pool 2 contained five (5) patients positive for the e14a2 variant. Both pools were diluted with the same negative pool containing 9 BCR-ABL negative patients.

Samples were diluted to an Expected value of 4.7 and anchored to the WHO-IS with Expected values of MR3.0 (0.1%IS) and MR4.0 (0.01%IS), and a College of American Pathologists (CAP) Proficiency sample, MRD-01(2018) at MR4.4, nearer to the LoD.

All samples were tested according to the kit IFU. Samples were tested in replicates of twenty (20) per day for four (4) days by one (1) operator on one (1) QXDX™ AutoDG ddPCR system with two (2) independent kit lots (20 replicates. x 4 days x 2 lots x 1 instrument = 160 replicates per sample), Calibrator checks and controls met product acceptance criteria.

A nonparametric approach was used to identify the limit of detection (LoD). For each sample and lot combination the percentage of tests falling at or below the Limit of Blank (0 copies of BCR-ABL; 0.0000% IS) was determined. If the percentage of tests results at or below the LoB was ≤ 5%, then the LoD was the concentration of the test sample. The concentration of each sample was the median MR level of all results within each kit lot. The LoD was determined for each kit lot and sample combination. The LoD for the QXDx™ BCR-ABL %IS Kit was determined as the worst (least MR value) of the four median MR levels.

For the e13a2 sample, 98.1% (157/160) of tests were above the LoB, with a median MR level of 4.7. For the e14a2 sample, 99.4% (159/160) of tests were above the LoB, with a median MR level of 4.7. In all combinations, the worst (least) median MR value was MR4.7.

The calculated limit of detection by this model for two (2) lots and 2 samples was found to be 0.002% IS BCR-ABL or MR 4.7.

{18}------------------------------------------------

Kit, Calibrator and Control Stability (CLSI EP25-A)

a. Real-time Stability:

Reagent shelf life stability studies were conducted using 3 lots with testing ongoing at T0, T1, T2.5, T5, T11, T12, T19 and T25 months.

Three lots of the QXDx™ BCR-ABL %IS Kit, Lot D, Lot H and Lot I, which were stored at -15°C , the outer limit of a -20°C freezer. The Droplet Reader Oil and the Automated Droplet Generation Oil for Probes were stored at 30°C. At each specified time point, the kits were removed from the storage and tested per IFU to quantify the %BCR-ABL/ABL in the controls and contrived samples. The temperature of the freezers and incubators were monitored and recorded throughout the study.

To date, the BCR-ABL %IS kit lots along with the droplet generation and reader oils have met the acceptance criteria and are stable under the storage conditions at 12 months for Lot D and 5 months for Lot H and Lot I.

b. Freeze-thaw Stability

Three (3) samples were prepared for use in this study at approximately MR2. MR3 and MR4. The MR2 and MR4 samples were prepared by blending Meg01 cell line RNA (containing the e13a2 variant) and the K562 cell line (containing the e14a2 variant), with RNA extracted from HeLa cells (containing ABL only) in water. Poly-A RNA was added to each sample for stability and to adjust the MR2 and MR4 sample RNA concentration to approximately 100 ng/ µL. The MR3 sample was prepared by blending RNA extracted from twelve (12) BCR-ABL positive patient blood samples (containing both the e13a2 and the e14a2 variants) with RNA extracted from one (1) BCR-ABL negative patient sample and diluted in water to achieve an RNA concentration of approximately 100 ng/μL.

Four QXDx™ BCR-ABL %IS kits from one lot stored at -20oC were used in this study. With the exception of the QXDx™ iScript Advanced reverse transcriptase, all components of one (1) kit were fully thawed at ambient temperature for 15 minutes, all kit component caps were removed, components were held uncapped for 2 minutes, components were capped and materials were returned to -20oC for a minimum of 8 hours prior to the next temperature cycle. The QXDx™ iScript Advanced reverse transcriptase was thawed on an ice block. This cycle was repeated 1, 4 or 8 times. One kit (reference) remained frozen throughout the study duration.

{19}------------------------------------------------

All components of the QXDx BCR-ABL IS kit demonstrated stable performance for at least 5 freeze-thaw cycles.

Specimen Stability (Whole Blood Stability) ﻦ

One (1) BCR-ABL positive patient sample (< MR1) and three (3) BCR-ABL negative patient samples were collected in EDTA tubes and shipped to Bio-Rad within 24 hours of the draw date. Each sample was divided into 4 aliquots which was processed immediately or stored at 2-8oC for 1 or 2 additional days.

The positive blood sample was serially diluted with a negative blood sample creating dilutions spanning the clinical decision point at each day/timepoint to prevent hemolysis. Each dilution was divided into multiple aliquots and the RNA was extracted using the Promega Maxwell CSC RNA blood kit and instrument as per the manufacturer's instructions. Four (4) replicate extractions were conducted for dilutions 0 and 1. To increase power at the lower end, eleven (11) replicate extractions were conducted for dilutions 2 and 3.

Each extracted RNA sample replicate was tested using one (1) lot of the QXDx™ BCR-ABL %IS Ki, by one operator and on one QXDx™ AutoDG ddPCR system as per the kit IFU.

For each sample, the allowable range was calculated as the mean MR value obtained on the reference time point (Day 1) plus or minus 0.5 log. For the remaining time points, the mean MR value and the 95% CI were calculated for each dilution. Each time point and sample type was considered to pass if the confidence interval (CI) for each test sample fell entirely within the allowable range. All samples tested met the specification:

Dilution	Day	Use	n	Mean %IS Value	Mean MR Value	Lower 95% CIlimit	Upper 95% CIlimit	± 0.5 log AllowableRange	Pass/Fail
0	1	Reference	4	44.9645	0.35	0.31	0.39	0-0.85	-
	2	Test	4	19.8590	0.71	0.57	0.85		Pass
	3	Test	4	37.2260	0.43	0.39	0.47		Pass
1	1	Reference	4	7.6568	1.12	1.07	1.16	0.62-1.62	-
	2	Test	4	3.7152	1.44	1.27	1.62		Pass
	3	Test	4	6.5420	1.18	1.15	1.22		Pass
2	1	Reference	11	0.7302	2.14	2.11	2.17	1.64-2.64	-
	2	Test	11	0.6424	2.19	2.17	2.22		Pass
	3	Test	11	0.6583	2.18	2.16	2.21		Pass
3	1	Reference	11	0.0511	3.32	3.20	3.42	2.82-3.82	-
	2	Test	11	0.0617	3.24	3.11	3.38		Pass

{20}------------------------------------------------

	10ﺎﺕ ﺍﻟﻤﺘﺤﺪﺓ ﺍﻟﻤﺘﺤﺪﺓ ﺍﻟﻤﺘﺤﺪﺓ ﺍﻟﻤﺘﺤﺪﺓ ﺍﻟﻤﺘﺤﺪﺓ ﺍﻟﻤﺘﺤﺪﺓ ﺍﻟﻤﺘﺤﺪﺓ ﺍﻟﻤﺘﺤﺪﺓ ﺍﻟﻤﺘﺤﺪﺓ ﺍﻟﻤﺘﺤﺪﺓ ﺍﻟﻤﺘﺤﺪﺓ ﺍﻟﻤﺘﺤﺪﺓ ﺍﻟﻤﺘﺤﺪﺓ ﺍﻟﻤﺘﺤﺪﺓ ﺍﻟﻤﺘﺤﺪﺓ ﺍﻟﻤﺘﺤﺪﺓ ﺍﻟﻤﺘﺤﺪﺓ ﺍﻟﻤﺘﺤﺪﺓ ﺍﻟﻤﺘﺤﺪﺓ ﺍﻟﻤﺘﺤﺪﺓ ﺍﻟﻤﺘﺤﺪﺓ ﺍﻟﻤ		0 0460U.UUUU	0 00ﺎ ﻙ ، ﻙ ، ﻙ ، ﻙ ، ﻙ ، ﻙ ، ﻙ ، ، ﻙ ، ، ، ، ، ، ، ، ، ، ، ، ، ، ، ، ، ، ، ، ، ، ، ، ، ، ، ، ، ، ، ، ، ، ، ، ، ، ، ، ، ، ، ، ، ، ، ، ، ، ، ، ، ، ، ، ، ، ، ، ، ، ، ، ، ، ، ، ، ،	0 40ر	0 00
--	--------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------	--	------------------	---------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------	-----------	------	--	--

Verification of WHO Standard Quantification

WHO-IS primary reference standards were run as patient samples on seven kit lots. The %IS master calibration CF, 0.93, was applied when the calibrator check values on each plate fell within allowable manufacturing specifications.

Four (4) levels of WHO primary standards were prepared and tested as patient samples as described in the QXDx™ BCR-ABL %IS kit IFU. The WHO primary standard cells were reconstituted in 600ul Qiagen RLT buffer, lysed using repeated pipette aspiration and RNA was extracted using the Qiagen RNeasy Mini Kit per NIBSC IFU. Each level was tested in four (4) replicates per the QXDx™ BCR-ABL %IS Kit IFU.

In all cases, the calibrator checks fell within their allowable ranges validating each of the runs. The WHO primary standard measured values as based on the master calibration curve and associated %IS correction factor (CF) were compared against the WHO assigned values via regression analyses. In all cases, the WHO standards measured within the allowable range of their actual assigned values from WHO:

		Slope (m)		Correlation (r2)			Intercept (b)
Bio-Rad KitLot	Allowable Range	Measured	Status	Allowable Range	Measured	Status	Allowable Range	Measured	Status
Lot A	0.95 –1.05	1.025	Pass	0.98 –1.00	0.997	Pass	-0.2 – 0.2	-0.045	Pass
Lot B		1.025	Pass		0.996	Pass		-0.045	Pass
Lot C		0.972	Pass		0.997	Pass		0.053	Pass
Lot F		1.026	Pass		0.992	Pass		-0.001	Pass
Lot G		0.967	Pass		0.995	Pass		0.090	Pass
Lot H		1.02	Pass		0.998	Pass		-0.005	Pass
Lot I		1.002	Pass		0.997	Pass		0.029	Pass
Overall		1.014	Pass		0.995	Pass		-0.006	Pass

Carryover Contamination

A high positive sample pool was prepared by diluting RNA extracted from the MEG-01 cell line, which expresses the e13a2 variant at a BCR-ABL/ABL ratio

{21}------------------------------------------------

measuring MR0.2, diluted in commercially available DNA suspension buffer. A negative sample consisting of nuclease-free water was also tested.

The QXDx™ DR reads droplets column wise from the top to bottom. To examine for possible carryover, two (2), ninety-six (96) well plates were set up with high positive (Hi Pos) and negative (Neg) wells in alternating rows. A total of four (4) rows each containing twelve (12) replicates or possible 48 carry over events were tested per plate. Two plates were tested on each of 3 QXDx™ DR instruments for a total test sample size of 288. Testing was conducted per the QXDxTM BCR-ABL %IS Kit IFU.

Of the 288 replicate carryover tests, two-hundred and eighty-six (286) wells met inclusion criteria. Two (2) wells were excluded due to unacceptable droplet event counts. Instrument operation was verified by % droplet occupancy which demonstrates positive sample was distributed amongst droplets. Occupancy rate was >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)

{22}------------------------------------------------

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.

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).