Predicate Devices

Reference Devices

Not Found

AI/ML (Artificial Intelligence / Machine Learning)

No
The summary describes a standard digital PCR assay and analysis based on Poisson distribution, with no mention of AI or ML in the device description, intended use, or performance studies. The software mentioned is for system control and result analysis, not for AI/ML-driven interpretation.

Therapeutic

No.
This device is an in vitro diagnostic (IVD) device used for monitoring treatment effectiveness in CML patients by quantifying specific gene transcripts, not for direct therapeutic intervention or treatment.

Diagnostic

No

Explanation: The "Intended Use / Indications for Use" section explicitly states, "This test is not intended for the diagnosis of CML." Instead, it is used for "monitoring of treatment with Tyrosine Kinase Inhibitors (TKIs)" in already diagnosed patients.

SaMD (Software as a Medical Device)

No

The device is a kit used with a physical instrument (Sniper Digital PCR All-in-One System) to perform a laboratory test. While it includes software for control and analysis, it is not solely software.

IVD (In Vitro Diagnostic)

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

Here's why:

Intended Use: The "Intended Use / Indications for Use" section explicitly states that the kit is an "in vitro nucleic acid amplification test" for the quantitation of specific transcripts in total RNA from whole blood. This clearly indicates that the test is performed outside of the body on biological samples.
Device Description: The description details the process of extracting RNA from peripheral blood and performing a "RT-dPCR (Reverse Transcription-Droplet PCR) reaction solution" using the system. This is a laboratory-based diagnostic procedure.
Sample Type: The test uses "total RNA from whole blood," which is a biological specimen.
Purpose: The purpose is to measure BCR-ABL1 to ABL1 levels for monitoring treatment in CML patients. This is a diagnostic application used to inform clinical decisions.

All of these points align with the definition of an In Vitro Diagnostic device.

PCCP (Predetermined Change Control Plan) Authorized

N/A

Overview

Intended Use / Indications for Use

The BCR-ABL1 (p210) %IS Kit (Digital PCR Method) 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) adult patients expressing BCR-ABL1 fusion transcripts type e13a2 and/or e14a2. The BCR-ABL1 (p210) %IS Kit (Digital PCR Method) is a reverse transcription-quantitative PCR performed on the Sniper Digital PCR All-in-One 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 BCR-ABL1 (p210) %IS Kit (Digital PCR Method) is intended for use only on the Sniper Digital PCR All-in-One 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 (comma separated list FDA assigned to the subject device)

OYX, PHG

Device Description

The BCR-ABL1 (p210) %IS Kit (Digital PCR Method) is designed for detection of the BCR-ABL1 fusion gene (p210) and ABL1 gene, with specific primers and specific fluorescence probes. The test process includes three parts. The first part is to extract ribonucleic acid (RNA) from peripheral blood of CML patients. The second part is to detect BCR-ABL1 fusion gene (p210) and ABL1 internal reference gene in RNA samples by RT-dPCR (Reverse Transcription-Droplet PCR) reaction solution using the Sniper Digital PCR All-in-One System (DQ24-Dx). The third part is to analyze the results.

The Sniper Digital PCR All-in-One System divides the sample into about 20000 droplets and carries out PCR amplification, read the number of positive and negative droplets through fluorescent signals, and then calculate the concentration of nucleic acid quantitatively according to the volume of the droplets and the principle of Poisson Distribution.

DQ24-Dx-Sight Software (v1.0.2) is used to control the system and analyze test results. This software is embedded in the Sniper Digital PCR All-in-One System.

Mentions image processing

Not Found

Mentions AI, DNN, or ML

Not Found

Input Imaging Modality

Not Found

Anatomical Site

Not Found

Indicated Patient Age Range

adult patients

Intended User / Care Setting

Prescription Use

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 (study type, sample size, AUC, MRMC, standalone performance, key results)

Precision/Reproducibility:

Sample Size: 540 observations for site precision, 864 observations for batch precision.
Study Design:
- 3 positive pools (e13a2, e14a2, mixed) at 5 concentrations (MR1.0, MR2.0, MR3.0, MR4.0, MR4.5) were diluted using a negative pool.
- Site Precision: Samples assayed in 2 replicates per run for 2 runs per day for 3 nonconsecutive days (1st, 3rd, 5th) at 3 sites (one instrument each) with one reagent lot. Total of 36 replicates per sample condition. Performed by 2 operators per site.
- Batch Precision: Samples assayed in 3 replicates per run for 2 runs per day for 3 nonconsecutive days (1st, 3rd, 5th) at 1 site (2 instruments) with 3 reagent lots. Total of 108 replicates per sample condition. Performed by 2 operators.
Key Results:
- Acceptance criteria for CV% (relative to MR value) were met for all concentrations and types of precision (within-run, within-day, between-day, within-site, between-site, total).
- MR levels showed low variability, including between sites and between lots.
- Calibrator and control precision also met acceptance criteria.

RNA Extraction Method:

Sample Size: 180 results
Study Design: 3 positive pools (e13a2, e14a2, K562 cells) at 5 concentrations (MR0.5-1.0 to MR4.2-4.5) prepared from peripheral blood. Samples pretreated on Day 1 by two operators using Whole Blood Nucleic Acid Extraction Reagent. RNA stored at -70℃. Samples were extracted twice by 2 operators per day for 3 nonconsecutive days with 1 reagent lot.
Key Results: 97% of extractions met RNA quality control (concentration ≥100 ng/μL, OD260/OD280 ratio > 1.6). For qualified RNA samples, CV% of all samples were less than 10%.

Linearity/Assay Reportable Range:

Sample Size: Not explicitly stated as N, but 10 levels for each of 2 variants, with 4 replicates each, so 80 assays.
Study Design: 2 positive pools (e13a2, e14a2) at 10 concentrations (MR0.3 to MR4.7) diluted with a negative pool. Samples assayed in 4 replicates for one day at 1 instrument with 1 reagent lot.
Key Results:
- Precision of all samples met the requirement of ≤10%.
- Deviation analysis from weighted least squares (WLS) linear regression showed % deviations of all samples met requirement of ≤±15%.
- R2 values for e13a2 and e14a2 linear range regression were 0.996 and 0.994 respectively, meeting the ≥0.98 requirement.
- Slope for e13a2 was 1.000 (95% CI: 0.98-1.02) and for e14a2 was 1.004 (95% CI: 0.98-1.03), both within the 0.83-1.20 acceptable range.
- The assay reportable range is 50%IS (MR0.3) - 0.0032%IS (MR4.5).

Traceability:

Study Design: Demonstrated by measuring the WHO Reference Panel with 9 independent kit lots. Each of 4 WHO Reference Panel members tested in 6 replicates across 9 runs (1 run per lot).
Key Results: Correlation with R2 values of 0.989-0.997. Slopes varied between 0.889 and 0.997, and intercepts between 0.011 and 0.222.

Detection Limit (Limit of Blank, Limit of Detection/Quantitation):

Limit of Blank (LoB): Tested 144 independent BCR-ABL negative samples. 138 had no detectable values. 6 had measurements below LoD and were reported as "MR value > 4.5". LoB is 0 copy.
Limit of Detection (LoD)/Limit of Quantitation (LoQ): Assessed using 2 positive pools (e13a2, e14a2) at 3 concentrations (MR4.5, MR4.7, MR4.9). Samples assayed in 20 replicates per day for 3 days with 2 reagent lots (total 120 replicates).
Key Results:
- MR4.5 samples: 100% hit rate, precision between 3.47% and 4.03%.
- MR4.7 samples: 97%-98% hit rate, precision between 4.09% and 4.79%.
- MR4.9 samples: 78%-90% hit rate, precision between 4.09% and 4.64%.
- The studies support an LoD of 4.7 and LoQ of 4.5 for the assay.

Analytical Specificity (Interference):

Study Design: CML positive patient samples (MR ~3.0) with potential interfering substances (cholesterol, bilirubin, EDTA, hemoglobin, heparin, triglycerides, etc.) added at recommended concentrations. Control group with diluent. 2 replicate extractions per sample, 3 replicates per extracted sample (total 6 tests).
Key Results: For MR values, mean test MR value and 95% CI fell within 95% CI ±0.5Log of control. For %IS data, 95% CI of mean %IS for test samples intersected detected range of control. All interferences passed acceptance criteria.

Analytical Specificity (Primer Specificity):

Study Design: Tested with p190 (e1a2), p230 (e19a2) samples at 4 dilutions, and p210 (e13a2, e14a2) samples at 4 MR levels.
Key Results:
- For p190 and p230 samples, negative specificity was 100% (expected ≥95%).
- For p210 samples, positive specificity was 100% and CV% met requirements (≤10%).

Analytical Specificity (Carryover Contamination):

Study Design: High positive and negative samples in alternating rows on 8-well PCR plates. 2 plates on each of 2 instruments (total 64 test samples).
Key Results: No signal was measured in the 32 negative wells. Acceptance criteria met, demonstrating no significant carryover.

RNA Input:

Study Design: 2 positive pools (e13a2, e14a2) at 4 MR levels (MR1.0, MR2.0, MR3.0, MR3.5). RNA input controlled at 30 ng, 150 ng, 300 ng, 500 ng, 800 ng, and 1000 ng. Assayed in 3-5 replicates per run with 1 reagent lot.
Key Results: At 500ng RNA input, positive detection rate was 100%, deviation between measured and theoretical values were within ±0.5, and precision were ≤10%. Therefore, 500ng RNA input is set for the kit.

Stability Studies (Real-Time Stability):

Study Design: 1 positive pool at 3 levels (MR2.0, MR3.0, MR4.0). 3 lots tested at T0, T3, T6, T9, T11, T12, T13.
Key Results: Kit performance met acceptance criteria up to T13, supporting a 12-month shelf life at -20°C±5°C. Measured MR values and 95% CI were within ±0.5 log of T0 results. Precision of measured MR values was between 0.56% and 5.95%.

Stability Studies (Freeze-thaw Stability):

Study Design: Kit components subjected to 3, 5, and 6 freeze-thaw cycles. Tested with same samples as real-time stability (MR2.0, MR3.0, MR4.0), controls, and calibrators.
Key Results: Stable performance for at least 5 freeze-thaw cycles. Measured MR values and 95% CI were within ±0.5 log of 0 time result. Precision was between 0.83% and 5.95%.

Specimen Stability (Peripheral Blood):

Study Design: 3 fresh peripheral blood samples from BCR-ABL1 positive patients (p210(e14a2), MR 0.3, 3.0, 4.0), stored at 2-8°C. RNA extracted on days 0, 1, and 2 after collection. Each extracted RNA tested 6-8 replicates using one kit lot.
Key Results: Peripheral blood samples stored for 1 day at 2-8°C are stable and produce reliable results. Measured MR values and 95% CI were within ±0.5 log of Day 0 result. Precision was between 0.92% and 5.75%.

Method Comparison with Predicate Device:

Study Type: Method comparison study
Sample Size: 112 clinical samples (originally 127, 15 excluded) obtained from individuals previously diagnosed with t(9;22) positive CML (p210). Samples covered MR values between 0.32 and 4.47.
Key Results:
- Passing-Bablok regression: Intercept A (95% CI): 0.17 (0.13-0.22), slope B (95% CI): 0.99 (0.97-1.01).
- Spearman correlation coefficient: 0.988 (P

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

September 5, 2023

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 the FDA logo, which 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.

Suzhou Sniper Medical Technologies Co., Ltd % Huifang Zhao Consultant Sinow Medical AS Hoyteknologisenteret. Thormohlens gate 55 Bergen, 5006 Norway

Re: K221869

Trade/Device Name: BCR-ABL1 (p210) %IS Kit (Digital PCR Method) Regulation Number: 21 CFR 866.6060, 21 CFR 862.2570 Regulation Name: BCR-ABL quantitation test Regulatory Class: Class II Product Code: OYX, PHG Dated: April 3, 2023 Received: April 4, 2023

Dear Huifang Zhao:

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

1

and listing (21 CFR Part 807); labeling (21 CFR Part

Page 2

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 803) for devices or postmarketing safety reporting (21 CFR 4, Subpart B) for combination products (see https://www.fda.gov/combination-products/guidance-regulatory-information/postmarketing-safety-reportingcombination-products); good manufacturing practice requirements as set forth in the quality systems (OS) 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 https://www.fda.gov/medical-device-safety/medical-device-reportingmdr-how-report-medical-device-problems.

For comprehensive regulatory information about mediation-emitting products, including information about labeling regulations, please see Device Advice (https://www.fda.gov/medicaldevices/device-advice-comprehensive-regulatory-assistance) and CDRH Learn (https://www.fda.gov/training-and-continuing-education/cdrh-learn). 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 (https://www.fda.gov/medical-device-advice-comprehensive-regulatoryassistance/contact-us-division-industry-and-consumer-education-dice) for more information or contact DICE by email (DICE@fda.hhs.gov) or phone (1-800-638-2041 or 301-796-7100).

Sincerely,

Pamela S. Ebrahimi -S Digitally signed by Pamela S. Ebrahimi -S Date: 2023.09.05 15:40:06 -04'00'

Pamela Gallagher Ebrahimi, Ph.D. Deputy Branch Chief Division of Molecular Genetics and Pathology OHT7: Office of In Vitro Diagnostics Office of Product Evaluation and Quality Center for Devices and Radiological Health

Enclosure

2

Indications for Use

510(k) Number (if known) K221869

Device Name

BCR-ABL1 (p210) %IS Kit (Digital PCR Method)

Indications for Use (Describe)

The BCR-ABL1 (p210) %S Kit (Digital PCR Method) 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) adult patients expressing BCR-ABL1 fusion transcripts type e13a2 and/or e14a2. The BCR-ABL1 (p210) %IS Kit (Digital PCR Method) is a reverse transcription-quantitative PCR performed on the Sniper Digital PCR All-in-One 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 BCR-ABL1 (p210) %IS Kit (Digital PCR Method) is intended for use only on the Sniper Digital PCR All-in-One 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)
-------------------------------------------------	--

X Prescription Use (Part 21 CFR 801 Subpart D)

Over-The-Counter Use (21 CFR 801 Subpart C)

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510(k) Summarv

A. Submitter:

Submitter: Suzhou Sniper Medical Technologies Co., Ltd.

Address: Unit 301, Building A6, Suzhou BioBAY, No. 218 Xinghu Street, Suzhou

Industrial Park, Suzhou Area, China (Jiangsu) Pilot Free Trade Zone, Suzhou,

China.

Email: zhangpeng@sniper-tech.com

Contact Person: Peng Zhang

B. Device

Trade Name:	BCR-ABL1 (p210) %IS Kit (Digital PCR Method)
Common Name:	BCR-ABL1 Digital PCR Test
Type of Test	Reverse transcription, quantitative, digital droplet
polymerase chain reaction (ddPCR) based nucleic acid
amplification
Regulation section:	21 CFR 866.6060
21 CFR 862.2570
Classification:	Class II
Product Code:	OYX
PHG
Classification/Advisory Panel	Pathology (88)

C. Predicate device:

Product Name: QXDx BCR-ABL %IS Kit for use on the QXDx AutoDG ddPCR System 510(k) Number: K181661 Product Code: OYX, PHG Manufacture: Bio-Rad Laboratories, Inc.

D. Indications for Use:

1. Indications for use:

The BCR-ABL1 (p210) %IS Kit (Digital PCR Method) 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) adult patients expressing BCR-ABL1 fusion transcripts type e13a2 and/or e14a2. The BCR-ABL1 (p210) %IS Kit (Digital PCR Method) is a

4

reverse transcription-quantitative PCR performed on the Sniper Digital PCR Allin-One 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 BCR-ABL1 (p210) %IS Kit (Digital PCR Method) is intended for use only on the Sniper Digital PCR All-in-One 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.

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

For prescription use only.

1. Special instrument requirements:
  Sniper Digital PCR All-in-One System

E. Device Description:

The BCR-ABL1 (p210) %IS Kit (Digital PCR Method) is designed for detection of the BCR-ABL1 fusion gene (p210) and ABL1 gene, with specific primers and specific fluorescence probes. The test process includes three parts. The first part is to extract ribonucleic acid (RNA) from peripheral blood of CML patients. The second part is to detect BCR-ABL1 fusion gene (p210) and ABL1 internal reference gene in RNA samples by RT-dPCR (Reverse Transcription-Droplet PCR) reaction solution using the Sniper Digital PCR All-in-One System (DQ24-Dx). The third part is to analyze the results.

A description of the reagents provided with the kit is described below in Table 1.

Composition	Main components	Application
One-step RT-dPCR
Master Mix	One-step RT-dPCR Buffer,
dNTP/dUTP Mix, MgCl2,
FAM Reference Dye,
RNase Inhibitor, etc.	Reaction mix component of the RT
reaction to generate cDNA from RNA
template.

Table 1: Reagents in the BCR-ABL1 (p210) %IS Kit (Digital PCR Method)

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Composition	Main components	Application
BCR-ABL1 Primer
Probe Mix	Primers, Probes	Provides primers and probes for ddPCR
amplification and detection of target
sequences.
BCR-ABL1
Enzyme Mix	Taq DNA Polymerases,
Reverse Transcriptase,
RNase Inhibitor, Uracil-
DNA Glycosylase	Catalyzes the amplification of primers
hybridized to templates from the cDNA.
Enzyme exonuclease activity degrades
hybridized probes to release fluorescence
for the detection of amplicons in each
PCR cycle.
Calibrator 10%IS	K562 cell RNA, HL60 cell
RNA mixture	Per run calibrators to check against
acceptance criteria for use of electronic
WHO-IS CF factor and reporting of
WHO-IS value results
Calibrator 0.1%IS	K562 cell RNA, HL60 cell
RNA mixture	Per run calibrators to check against
acceptance criteria for use of electronic
WHO-IS CF factor and reporting of
WHO-IS value results
Positive Control 1
(%IS of 10)	K562 cell RNA, HL60 cell
RNA mixture	Control used to ensure that ddPCR steps
performed properly by generating expected
MR value.
Positive Control 2
(%IS of 0.01)	K562 cell RNA, HL60 cell
RNA mixture	Control used to ensure that ddPCR steps
performed properly by generating expected
MR value.
Negative Control	HL60 cell RNA	Negative control used to ensure that RT
and ddPCR steps performed properly and
identify false positive results due to
contamination.
Nuclease free water	DNase/RNase-Free water	Adjust volume of RT & ddPCR
reactions.

Instrument: _

The Sniper Digital PCR All-in-One System consists of one instrument, which can be used together with it's supporting consumables and BCR-ABL1 (p210) %IS Kit (Digital PCR Method) to complete the detection of samples.

The Sniper Digital PCR All-in-One System divides the sample into about 20000 droplets and carries out PCR amplification, read the number of positive and negative droplets through fluorescent signals, and then calculate the concentration of nucleic acid quantitatively according to the volume of the droplets and the principle of Poisson Distribution.

Software: _

DQ24-Dx-Sight Software (v1.0.2) is used to control the system and analyze test results. This software is embedded in the Sniper Digital PCR All-in-One System.

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F. Substantial Equivalence Information:

Predicate device name(s):

The QXDx BCR-ABL %IS Kit

QXDx Automated Droplet Generator

QXDx Droplet Reader

QXDx Software 1.2

2.Predicate 510(k) number(s):

K181661

3. Comparison with predicate:

	Similarities
Item	Subject Device	Predicate Device
Indications	The BCR-ABL1 (p210) %IS Kit
(Digital PCR Method) 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)
adult patients expressing BCR-ABL1
fusion transcripts type e13a2 and/or
e14a2. The BCR-ABL1 (p210) %IS
Kit (Digital PCR Method) is a reverse
transcription-quantitative PCR
performed on the Sniper Digital PCR
All-in-One 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 BCR-ABL1 (p210) %IS Kit
(Digital PCR Method) is intended for
use only on the Sniper Digital PCR
All-in-One 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.	The QXDxTM 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 QXDxTM
AutoDGTM 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 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.
Measurement
Type	Quantitative	Same
Specimen
Type	RNA from whole blood (EDTA)	Same
Anti-coagulant	EDTA	Same

Table 2: Similarity comparison

7

Similarities
Item	Subject Device	Predicate Device
Traceability	1st WHO International Genetic
Reference Panel for quantitation of
BCR-ABL translocation by RQ-PCR	Same
Reporting
Units	Both %IS and Molecular Response
(MR)	Same
Fundamental
Technology	Digital PCR	Same
Calibrators	Two levels are formulated at 0.1%IS
and 10%IS BCR-ABL1/ABL1.	Same

Table 3: Different comparison

	Differences
Item	Subject Device	Predicate Device
Measuring
Range	MR 0.3 to MR 4.5	MR 0.3 to MR 4.7
RNA Input	500 ng	1000n g
Quality
calibrators	3 levels of external control
Positive control 1 (%IS of 10)
Positive control 2 (%IS of 0.01)
Negative Control	3 levels of external control
RNA High (%IS of 18)
RNA Low (%IS of 0.03)
RNA Negative
Instrument	Sniper DQ24-Dx	Bio-Rad QXDXTM AutoDGTM
ddPCR System
Instrument
Computer
Operating
System	Embedded software, Ubuntu18.04.5	Microsoft Windows 10
Degree of
Automation	Same. Automated control of amplification,
detection, and data analysis.	Requires manual transfer of
amplification mixture to
amplification/detection instrument.
Automated control of detection and
data analysis, except amplification
functionality.
Amplification
Reaction
Volume	22 µL in Sniper PCR plates.	20-25 µL in 96-well Bio- Rad PCR
plates.

G. Standard/Guidance Document Referenced (if applicable):

CLSI EP07-Ed3, Interference Testing in Clinical Chemistry

CSLI EP17-A2, Evaluation of Detection Capability for Clinical Laboratory

Measurement Procedures.

CSLI EP15-A3, User Verification of Precision and Estimation of Bias- Third Edition.

CLSI EP06-2nd Edition, Evaluation of the Linearity of Quantitative Measurement Procedures.

CLSI EP25-A Evaluation of Stability of In Vitro Diagnostic Reagents.

8

H. Test Principle:

The BCR-ABL1 (p210) %IS Kit quantitatively detects the RNA of fusion genes BCR-ABL1 (p210, b2a2 (e13a2) and b3a2 (e14a2)) and ABL1 in the peripheral blood of adult patients with Chronic Myeloid Leukemia (CML) by designed specific primers and probes combined with Sniper Digital PCR All-in-One System.

Total RNA is extracted from whole blood containing EDTA anticoagulant for detection. Sample RNA is mixed with One-step RT-dPCR Master Mix, BCR-ABL1 Primer Probe Mix and BCR-ABL1 Enzyme Mix to prepare a 22 µL PCR reaction. The BCR-ABL1 primers and probes are designed to detect the breakpoint translocation of BCR-ABL1 p210 [b2a2 (e13a2) and b3a2 (e14a2)], and to detect the ABL1 sequence.

A total of 22 uL of PCR reaction is loaded into each of 8 consecutive tubes, which is placed on the sample rack of the Sniper Digital PCR All-in-One System. The detection is conducted directly without separated reverse transcription. The supporting consumables required for the detection process include droplet generation oil, PCR four-well plate, droplet generation needle, 4 quality controls and 2 calibrators for each run. After amplification, Sniper Digital PCR All-in-One System will photograph each sample, control and calibrator according to the fluorescence channels to distinguish negative and positive droplets, and the photos are stored.

After the detection is completed, set the threshold line at 1/3 of the distance between negative and positive droplet clusters, and the ratio of BCR-ABL1 and ABL1 genes is calculated, and the %IS value and MR value of the sample are calculated according to the conversion factor (CF) of the kit. Secondly, the quality of the test results should be controlled, in which the blank control should satisfy BCR-ABL1 copy ≤1 and ABL1 copy ≤10, the negative control should satisfy BCR-ABL1 copy ≤1, the measured values of positive control 1 and calibrator 10%IS should be between MR0.5 and MR1.5, the measured value of positive control 2 should be between MR3.5 and MR4.5, and the measured value of calibrator 05-6

9

0.1%IS should be between MR2.5 and MR3.5. Finally the results are outputted. 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 repetitive detection of a sample is non-normally distributed about the mean. %IS values require log transformation prior to performing any statistical analyses that require normally-distributed data. Another value commonly reported in the literature is the Molecular Reduction, or MR value. The MR value is traditionally written as MRx.x. However, for simplicity and legibility, the BCR-ABL1 (p210) %IS Kit (Digital PCR Method) 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 shown below table 4:

%IS	MR
50	0.3
32	0.5
10	1.0
1	2.0
0.32	2.5
0.1	3.0 (MMR)
0.032	3.5
0.01	4.0
0.0032	4.5
0.001	5.0
MMR: Major Molecular Response

Table 4: %IS and MR comparison table

The results are interpreted automatically by the embedded Software DQ24-Dx-Sight from measured droplet counts, fluorescent signals, and embedded calculation algorithms. It will report out BCR-ABL1 and ABL1 copies. An indication of sample suitability is that the ABL1 copies are sufficient for the

10

MRx.x column. International Scale Percent Ratio (%IS) is calculated as the copy number of BCR-ABL1 divided by the copy number of ABL1, then multiplied by 100 times the conversion factor (CF) of the kit, i.e.

%IS=BCR-ABL1copy / ABL1copy ×100 × CF

The test results should be interpreted according to the following standards (table5):

Test results	Report results	Explanation of test results
Copy number of BCR-
ABL1 as 0.	Report: Negative	It indicates that there is no BCR-ABL1
fusion gene in the test sample.
MR > 4.5 or %IS 4.5
detected or %IS value 140,000 and the BCR-ABL1 copy (3) number > 0, the ABL1 copy number exceeds the linear range, which will affect the quantitative accuracy. A retest should be performed with reduced RNA input.

Performance Characteristics (if/when applicable): L.

Analytical Performance:

a. Precision/Reproducibility:

Precision and Reproducibility were assessed using 3 positive pools at 5 levels. The 3 positive pools were prepared by mixing 5 BCR-ABL1 positive p210(e13a2) RNA samples with an MR value of 0.3 (pool 1), 5 BCR-ABL1

11

positive p210(e14a2) RNA samples with an MR value of 0.3 (pool 2), and 5 BCR-ABL1 positive RNA samples (p210(e13a2) and p210(e14a2) with an MR value of 0.3) (pool 3). A negative pool was used as a diluent and was prepared by mixing 30-60 BCR-ABL1 negative RNA samples with a ratio value of 0%. The positive sample pools were diluted with the negative sample pool to generate five samples with different concentrations: MR1.0, MR2.0, MR3.0, MR4.0 and MR4.5. Each sample was stored separately at the temperature of -20°C±5°C.

Samples were assayed in 2 replicates per run for 2 runs per day for 3 nonconsecutive days (1st, 3rd and 5th) at 3 sites (one instrument at each site) with one reagent lot for a total of 36 replicates. Each run was performed by an independent operator (2 operators per site). The total precision (CV, %) values were required to meet the requirements shown in the following table 6. Table 6: Precision requirements corresponding to different concentrations

MR value	Precision (CV, %) requirements
MR0.3-MR2.0	$\leq$ 10%
MR2.1-MR3.49	$\leq$ 15%
MR3.5-MR4.0	$\leq$ 20%
LOQ	$\leq$ 20%

A total of 540 observations were included in a variance components analysis with random effects for site, day, and run (operator) to assess repeatability, within-day precision, within-site precision and reproducibility of measured MR level. Results of MR level indicated low variability, including between sites, and all acceptance criteria were satisfied (CVs ≤10%). Total MR and %IS precision were calculated for the assay (see table 7 and table 8) and in-kit calibrators and controls (see table 9). The acceptance criteria were met.

Table 7: Precision analysis results (MR) of samples

| Sample | Variant | N | MR
expected | MR
mean | Within-run | | Within-day | | Between-day | | Within-site | | Between-site | | Total | |
|--------|---------|----|----------------|------------|------------|-------|------------|-------|-------------|-------|-------------|-------|--------------|-------|-------|-------|
| | | | | | SD | CV% | SD | CV% | SD | CV% | SD | CV% | SD | CV% | SD | CV% |
| MR1.0 | e13a2 | 36 | 1.00 | 1.05 | 0.001 | 0.06% | 0.006 | 0.58% | 0.012 | 1.12% | 0.012 | 1.12% | 0.016 | 1.57% | 0.019 | 1.85% |
| | e14a2 | 36 | 1.00 | 1.04 | 0.005 | 0.51% | 0.013 | 1.22% | 0.020 | 1.89% | 0.020 | 1.96% | 0.020 | 1.96% | 0.024 | 2.31% |
| | mix | 36 | 1.00 | 1.05 | 0.009 | 0.84% | 0.016 | 1.56% | 0.017 | 1.64% | 0.019 | 1.84% | 0.014 | 1.29% | 0.025 | 2.38% |
| MR2.0 | e13a2 | 36 | 2.00 | 1.99 | 0.008 | 0.40% | 0.013 | 0.66% | 0.023 | 1.13% | 0.024 | 1.20% | 0.023 | 1.14% | 0.036 | 1.82% |

12

| Sample | Variant | N | MR
expected | MR
mean | Within-run | | Within-day | | Between-day | | Within-site | | Between-site | | Total | |
|-----------------------------------------------------------|---------|----|----------------|------------|------------|-------|------------|-------|-------------|-------|-------------|-------|--------------|-------|-------|-------|
| | | | | | SD | CV% | SD | CV% | SD | CV% | SD | CV% | SD | CV% | SD | CV% |
| | e14a2 | 36 | 2.00 | 1.98 | 0.016 | 0.80% | 0.020 | 1.02% | 0.009 | 0.48% | 0.018 | 0.92% | 0.021 | 1.04% | 0.030 | 1.54% |
| | mix | 36 | 2.00 | 1.98 | 0.008 | 0.41% | 0.017 | 0.84% | 0.017 | 0.88% | 0.019 | 0.96% | 0.017 | 0.87% | 0.031 | 1.56% |
| | e13a2 | 36 | 3.00 | 2.99 | 0.014 | 0.47% | 0.032 | 1.06% | 0.007 | 0.24% | 0.016 | 0.52% | 0.027 | 0.91% | 0.071 | 2.37% |
| MR3.0 | e14a2 | 36 | 3.00 | 2.98 | 0.047 | 1.57% | 0.010 | 0.34% | 0.020 | 0.68% | 0.050 | 1.69% | 0.040 | 1.35% | 0.093 | 3.11% |
| | mix | 36 | 3.00 | 3.00 | 0.029 | 0.96% | 0.035 | 1.16% | 0.020 | 0.68% | 0.035 | 1.16% | 0.052 | 1.72% | 0.077 | 2.55% |
| MR4.0 | e13a2 | 36 | 4.00 | 3.92 | 0.057 | 1.45% | 0.044 | 1.12% | 0.023 | 0.60% | 0.061 | 1.55% | 0.041 | 1.05% | 0.135 | 3.43% |
| | e14a2 | 36 | 4.00 | 3.94 | 0.036 | 0.90% | 0.053 | 1.34% | 0.026 | 0.65% | 0.044 | 1.10% | 0.036 | 0.90% | 0.091 | 2.31% |
| | mix | 36 | 4.00 | 3.91 | 0.025 | 0.64% | 0.041 | 1.04% | 0.025 | 0.64% | 0.035 | 0.90% | 0.063 | 1.62% | 0.119 | 3.04% |
| | e13a2 | 36 | 4.50 | 4.66 | 0.049 | 1.05% | 0.055 | 1.19% | 0.091 | 1.95% | 0.103 | 2.21% | 0.194 | 4.18% | 0.255 | 5.48% |
| MR4.5 | e14a2 | 36 | 4.50 | 4.59 | 0.100 | 2.17% | 0.099 | 2.14% | 0.045 | 0.97% | 0.108 | 2.35% | 0.170 | 3.70% | 0.261 | 5.68% |
| | mix | 36 | 4.50 | 4.59 | 0.048 | 1.05% | 0.121 | 2.64% | 0.041 | 0.88% | 0.062 | 1.36% | 0.164 | 3.58% | 0.227 | 4.95% |
| Mix: Represents a mixture of e13a2 and e14a2 transcripts. | | | | | | | | | | | | | | | | |

Table 8: Precision analysis results (%IS) of samples

Sample	Variant	N	%IS expected	%IS mean	Within-run		Within-day		Between-day		Within-site		Between-site		Total
					SD	CV%	SD	CV%	SD	CV%	SD	CV%	SD	CV%	SD	CV%
MR1.0	e13a2	36	10.000	8.985	0.016	0.2%	0.123	1.4%	0.240	2.7%	0.241	2.7%	0.336	3.7%	0.397	4.4%
	e14a2	36	10.000	9.070	0.112	1.2%	0.270	3.0%	0.409	4.5%	0.424	4.7%	0.308	3.4%	0.503	5.5%
	mix	36	10.000	9.022	0.185	2.1%	0.327	3.6%	0.356	3.9%	0.400	4.4%	0.274	3.0%	0.507	5.6%
MR2.0	e13a2	36	1.000	1.017	0.019	1.9%	0.030	3.0%	0.053	5.2%	0.056	5.5%	0.054	5.3%	0.085	8.4%
	e14a2	36	1.000	1.051	0.038	3.6%	0.049	4.7%	0.022	2.1%	0.044	4.2%	0.050	4.8%	0.073	7.0%
	mix	36	1.000	1.048	0.020	1.9%	0.037	3.6%	0.042	4.0%	0.046	4.4%	0.040	3.8%	0.072	6.9%
MR3.0	e13a2	36	0.100	0.103	0.003	2.9%	0.008	7.4%	0.002	2.0%	0.004	3.5%	0.006	6.0%	0.017	16.3%
	e14a2	36	0.100	0.107	0.010	9.6%	0.002	2.3%	0.005	5.1%	0.012	10.8%	0.009	8.7%	0.022	20.9%
	mix	36	0.100	0.101	0.006	5.6%	0.007	7.0%	0.004	3.9%	0.007	6.8%	0.011	10.6%	0.016	15.8%
MR4.0	e13a2	36	0.010	0.013	0.002	12.1%	0.001	10.6%	0.001	5.2%	0.002	13.0%	0.001	9.2%	0.004	29.5%
	e14a2	36	0.010	0.012	0.001	7.6%	0.001	11.4%	0.001	5.6%	0.001	9.4%	0.001	8.4%	0.002	20.2%
	mix	36	0.010	0.013	0.001	5.4%	0.001	10.7%	0.001	6.1%	0.001	8.1%	0.002	16.1%	0.004	29.7%
MR4.5	e13a2	36	0.0032	0.0026	0.0001	5.8%	0.0004	16.3%	0.0004	16.5%	0.0004	17.5%	0.0010	40.3%	0.0015	56.6%
	e14a2	36	0.0032	0.0030	0.0004	14.7%	0.0008	25.3%	0.0004	13.8%	0.0006	20.0%	0.0009	31.2%	0.0015	51.3%
	mix	36	0.0032	0.0029	0.0003	9.9%	0.0007	23.1%	0.0002	5.3%	0.0003	11.1%	0.0010	33.6%	0.0013	44.8%

Table 9: Calibrator and Control Precision Analysis Results

sample	N	MR				%IS
		target	mean	SD	CV	target	mean	SD	CV%
Calibrators 10%IS	54	1.00	1.05	0.022	2.10%	10.00	8.861	0.443	5.00%
Calibrators 0.1%IS	54	3.00	3.01	0.054	1.79%	0.10	0.098	0.012	12.20%
Positive control 1	54	1.00	1.06	0.023	2.14%	10.00	8.766	0.454	5.18%
Positive control 2	54	4.00	3.91	0.105	2.68%	0.01	0.013	0.003	23.86%
Negative control	54	--	NA	NA	NA	--	0.000	0.000	NA
Blank control	54	--	NA	NA	NA	--	NA	NA	NA

13

Precision between batches

Precision between batches were assessed using 2 positive pools at 4 levels. The 2 positive pools were prepared by mixing 5 BCR-ABL1 positive p210(e13a2) RNA samples with an MR value of 0.3 (pool 1), and 5 BCR-ABL1 positive p210(e14a2) RNA samples with an MR value of 0.3 (pool 2). A negative pool was used as a diluent and was prepared by mixing 60 BCR-ABL1 negative RNA samples with a ratio value of 0%. The positive sample pools were diluted with the negative sample pool to generate four samples with different concentrations: MR1.0, MR3.0, MR4.0 and MR4.5. Each sample was stored separately at the temperature of -20°C±5°C.

Samples were tested in 3 replicates per run for 2 runs per day for 3 nonconsecutive days (1st, 3rd and 5th) at 1 site (2 instruments) with 3 reagent lots for a total of 108 replicates. Each run was performed by an independent operator (2 operators). The total precision (CV, %) values were required to meet the requirements shown in the following Table 10.

MR value	Precision (CV, %) requirements
MR0.3-MR2.0	1.6

Table 14: RNA quality control

16

| Variant | MR value extraction | | Sample detection | | | | |
|---------------------------------------------------------------------------------------------------|-----------|------- | | N | Qualified
N | Qualified
proportion | Tests
N | MR
SD | MR
Mean | CV, % |
| | MR0.5~~1.0 | 12 | | MR1.5~~2.0 | 12 | e13a2 | MR2.5~~3.0 | 12 | | MR3.5~~4.0 | 12 | | MR4.2~~4.5 | 12 | | MR0.5~~1.0 | 12 | | MR1.5~~2.0 | 12 | e14a2 | MR2.5~~3.0 | 12 | | MR3.5~~4.0 | 12 | | MR4.2~~4.5 | 12 | | MR0.5~~1.0 | 12 | K562 | MR1.5~~2.0 | 12 | | MR2.5~~3.0 | 12 | | MR3.5~~4.0 | 12 | | MR4.2~4.5 | 12 | *: Indicates that RNA concentration or purity does not meet requirements during extraction and no | | | Peripheral blood sample
--------------------------------|----------------|-------------------------|------------|----------|------------|-------|
| Sample
| 12 | | 12 | 0.016 | 0.81 | 2.04% |
| 12 | | 12 | 0.028 | 1.75 | 1.61% |
| 11* | | 11* | 0.043 | 2.50 | 1.70% |
| 12 | | 12 | 0.129 | 3.80 | 3.41% |
| 12 | | 12 | 0.246 | 4.51 | 5.44% |
| 10* | | 10* | 0.012 | 0.83 | 1.44% |
| 12 | | 12 | 0.023 | 1.75 | 1.33% |
| 12 | 97% | 12 | 0.040 | 2.52 | 1.58% |
| 12 | | 12 | 0.103 | 3.83 | 2.70% |
| 12 | | 12 | 0.320 | 4.63 | 6.91% |
| 12 | | 12 | 0.016 | 0.81 | 2.02% |
| 12 | | 12 | 0.033 | 1.74 | 1.88% |
| 10* | | 10* | 0.071 | 2.52 | 2.82% |
| 12 | | 12 | 0.139 | 3.76 | 3.71% |
| 12 | | 12 | 0.211 | 4.43 | 4.76% |
| | | | | | |

Table15: RNA extraction and detection

b. Linearity/Assay reportable range:

subsequent testing.

Linearity/Assay reportable range were assessed using 2 positive pools at 10 levels. The 2 positive pools were prepared by mixing 2 BCR-ABL1 positive p210(e13a2) RNA samples with an MR value of 0.3 (pool 1), and 2 BCR-ABL1 positive p210(e14a2) RNA samples with an MR value of 0.3 (pool 2). A negative pool was used as a diluent and was prepared by mixing 22 BCR-ABL1 negative RNA samples with a ratio value of 0%. The positive sample pools were diluted with the negative sample pool to generate ten samples with different concentrations: MR0.3, MR0.5, MR1.0, MR1.5, MR2.0, MR2.5,

17

MR3.0, MR4.0, MR4.5 and MR4.7. Each sample was stored separately at the temperature of 2-8°C.

Samples were assayed in 4 replicates for one day at 1 instrument with 1 reagent lot. The precision analysis and deviation analysis were required to meet the requirements shown in the following Table 16.

Category	Requirements
Precision	$\leq 10%$
% Deviation	$\leq \pm 15%$

Table 16: Precision and deviation requirements

Samples with concentrations from 50%IS (MR0.3) to 0.002%IS (MR 4.7) were determined for the e13a2 and e14a2 variants. Precision analysis showed that the precision of all samples met the requirement of ≤10%. Regression analysis showed that the appropriate type of regression analysis was a weighted least squares (WLS) linear regression analysis with no intercept (Y=AE). Based on WLS linear regression analysis, deviation analysis showed that the % deviations of all samples meet the requirement of ≤±15% (see table 17).

Variant	Sample	Precision				Acceptable range	Deviation				Pass
		Mean Y	Expected E	SD	CV %		Predicted y=AE	Deviation on	%Deviation on	Acceptable range
e13a2	1	0.26	0.30	0.009	3.26%	CV≤10%	0.30	-0.03	-10.91%	4.5". Based on nonparametric analysis method, the LoB of the BCR-ABL 1 (p210) %IS Kit (Digital PCR Method) kit is 0 copy.

Limit of Detection/ Limit of Quantitation

Limit of Detection/ Limit of Quantitation were assessed using 2 positive pools at 3 levels. The 2 positive pools were prepared by marking 1 BCR-ABL1 positive p210(e13a2) RNA sample with an MR value of 0.3 (pool 1), and 3 BCR-ABL1 positive p210(e14a2) RNA samples with an MR value of 0.3 (pool

20

2). A negative pool was used as a diluent and was prepared by mixing 39 BCR-ABL1 negative RNA samples with a ratio value of 0%. The positive sample pools were diluted with the negative sample pool to generate three samples with different concentrations: MR4.5, MR4.7 and MR4.9. Each sample was stored separately at the temperature of -20℃±5℃.

Samples were assayed in 20 replicates per day for 3 days with 2 reagent lots for a total of 120 replicates. Based on Probit method and Precision analysis, the LoD and LoQ of the kit can be obtained. The hit rate and precision were required to meet the requirements shown in the following Table 19.

Category	Hit rate	CV%
LoD	≥95%	None
LoQ	100%	≤10%

Table 19: Hit rate and precision requirements

The hit rate and precision of different samples were as follows. The hit rates of MR4.5 samples were 100%, the precision was between 3.47% and 4.03%. The hit rates of MR4.7 samples were between 97% and 98%, the precision was between 4.09% and 4.79%. The hit rates of MR4.9 samples were between 78% and 90%, the precision was between 4.09% and 4.64%. When the samples concentration was MR4.7, the hit rates could meet the requirement of ≥95%. When the samples concentration was MR4.5, the hit rates were 100% and the precisions were ≤10%. The studies support an LoD of 4.7 and LoQ of 4.5 for the assay.

e. Analytical specificity:

Interference

A sample pool was prepared by mixing peripheral blood from CML positive patients, with an MR value around 3.0. The potential interfering substance was added to the test group and diluent was added to the control group in concentrations recommended by CLSI EP7-A2. For both the control and test samples, 2 replicate extractions were performed and each extracted sample was

21

tested in replicates of 3 for a total of 6 tests per sample type.

For the MR values, the mean test MR value and 95% confidence interval needed to fall within the 95% confidence interval ±0.5Log of the corresponding control group. In all cases, samples passed the acceptance criteria (see table 20). For the %IS data, the 95% confidence interval of the mean %IS for test samples needed to intersect the detected range of the corresponding control group. In all cases, samples passed the acceptance criteria (see table 21). The potential interfering substances evaluated were cholesterol (6.47mmoL/L), conjugated bilirubin (86μM), EDTA (7mg/mL), hemoglobin (200g/L), sodium heparin (3000U/L), triglycerides (5.6mmoL/L), unconjugated bilirubin (257uM),10x red blood cell lysis buffer, phenol, residual ethanol, 10x PBS, and genomic DNA. Results demonstrated that these endogenous and exogenous substances tested did not impact test results as compared to control. However, considering that ethanol is an organic solvent that causes fusion of the droplets during amplification testing, residual ethanol in RNA samples should be minimized.

| Interfering substances | Mean value of MR | | 95% confidence
interval | | The acceptable range of
the AVG value of the
test and its 95%CI
(95% CI ±0.5 log of
control group) | Result (if
within
acceptable
range) |
|------------------------------------|------------------|---------------|----------------------------|---------------|----------------------------------------------------------------------------------------------------------------|----------------------------------------------|
| | Control
group | Test
group | Control
group | Test
group | | |
| Cholesterol | 3.04 | 2.98 | 2.94-3.13 | 2.94-3.01 | 2.44-3.63 | YES |
| Conjugated bilirubin | 3.01 | 2.99 | 2.97-3.06 | 2.94-3.03 | 2.47-3.56 | YES |
| EDTA | 3.00 | 3.00 | 2.97-3.04 | 2.93-3.08 | 2.47-3.54 | YES |
| Hemoglobin | 3.03 | 3.06 | 3.00-3.05 | 3.01-3.11 | 2.50-3.55 | YES |
| Heparin sodium | 3.06 | 3.02 | 2.99-3.14 | 2.98-3.06 | 2.49-3.64 | YES |
| Triglyceride | 2.98 | 3.07 | 2.93-3.04 | 3.05-3.08 | 2.43-3.54 | YES |
| Unconjugated bilirubin | 2.98 | 3.02 | 2.96-3.00 | 2.97-3.08 | 2.46-3.50 | YES |
| 10x Red blood cell lysis
buffer | 2.96 | 2.97 | 2.92-2.99 | 2.93-3.01 | 2.42-3.49 | YES |
| Phenol | 2.95 | 2.97 | 2.92-2.99 | 2.93-3.00 | 2.42-3.49 | YES |
| Ethanol | 2.95 | 2.95 | 2.91-2.99 | 2.92-2.97 | 2.41-3.49 | YES |
| 10x PBS | 2.97 | 3.03 | 2.92-3.03 | 2.90-3.16 | 2.42-3.53 | YES |

Table 20: Statistical of test results (MR values) of different interfering substances
---------------------------------------------------------------------------------------	--

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| Interfering substances | Mean value of MR | | 95% confidence
interval | | The acceptable range of
the AVG value of the | Result (if
within |
|------------------------|------------------|---------------|----------------------------|---------------|-------------------------------------------------------------|----------------------|
| | Control
group | Test
group | Control
group | Test
group | test and its 95%CI
(95% CI ±0.5 log of
control group) | acceptable
range) |
| Genomic DNA | 2.99 | 3.05 | 2.95-3.03 | 2.95-3.15 | 2.45-3.53 | YES |

Table 21: Statistical of test results (%IS values) of different interfering substances

	Mean value of %IS		%IS	Does the detected range
Interfering
substances	Control
group	Test
group	Detected range of
control group	%95 confidence
interval of the
detection	of control group
intersect with the
95%CI of the detection
Cholesterol	0.094%	0.106%	0.055%-0.112%	0.098%-0.114%	YES
Conjugated
bilirubin	0.098%	0.104%	0.088%-0.116%	0.093%-0.114%	YES
EDTA	0.100%	0.101%	0.089%-0.113%	0.083%-0.119%	YES
Hemoglobin	0.094%	0.088%	0.084%-0.102%	0.078%-0.098%	YES
Heparin
sodium	0.089%	0.096%	0.060%-0.114%	0.086%-0.105%	YES
Triglyceride	0.105%	0.086%	0.087%-0.125%	0.083%-0.088%	YES
Unconjugated
bilirubin	0.106%	0.096%	0.098%-0.115%	0.083%-0.108%	YES
10x Red blood
cell lysis buffer	0.111%	0.108%	0.098%-0.121%	0.098%-0.119%	YES
Phenol	0.112%	0.109%	0.097%-0.125%	0.100%-0.117%	YES
Ethanol	0.112%	0.114%	0.098%-0.134%	0.106%-0.121%	YES
10x PBS	0.108%	0.098%	0.080%-0.125%	0.074%-0.122%	YES
Genomic DNA	0.103%	0.091%	0.089%-0.115%	0.073%-0.110%	YES

Primer Specificity

Primer Specificity was assessed using 4 positive samples at 4 levels. 1 BCR-ABL1 positive p190(e1a2) RNA sample with a ratio of approximately 50%, 1 BCR-ABL1 positive p230(e19a2) RNA sample with a ratio of approximately 50%, 1 BCR-ABL1 positive p210(e13a2) RNA sample with an MR value of about 0.3, and 1 BCR-ABL1 positive p210(e14a2) RNA sample with an MR

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value of about 0.3. A negative pool was used as a diluent and was prepared by mixing 5 BCR-ABL1 negative RNA samples with a ratio value of 0%. The p190(e1a2) and p230(e19a2) samples were diluted with the negative sample pool to generate four samples with different concentrations: 50%, 10%, 0.1%, and 0.005%. The p210 samples were diluted with the negative sample pool to generate four samples with different concentrations: MR0.3, MR1.0, MR3.0 and MR4.5. Each sample was stored separately at the temperature of 2-8 °C. Samples were assayed in 4 replicates per run with 1 reagent lot. For p190 and p230 samples, the negative specificity should be ≥95%. For p210 samples, the positive specificity should be 100% and the CV% should be ≤10%. The test results showed that for p190 and p230 samples, the negative specificity

was 100%, for p210 samples, positive specificity and precision could meet the requirements. The results are shown in the Table 22 and Table 23 below.

| Variant | N | Ratio% | | | Acceptable
range | | Pass |
|-----------------|---|-------------------|------------------------|-------------------------------|-------------------------|-------------------------|------|
| | | Targeted
value | Detected mean
value | CV% of Detected
mean value | Negative
specificity | Negative
specificity | |
| p190
(e1a2) | 4 | 50% | 0.00% | 0.00% | 100 | ≥95% | YES |
| | 4 | 10% | 0.00% | 0.00% | 100 | | YES |
| | 4 | 0.1% | 0.00% | 0.00% | 100 | | YES |
| | 4 | 0.005% | 0.00% | 0.00% | 100 | | YES |
| p230
(e19a2) | 4 | 50% | 0.00% | 0.00% | 100 | ≥95% | YES |
| | 4 | 10% | 0.00% | 0.00% | 100 | | YES |
| | 4 | 0.1% | 0.00% | 0.00% | 100 | | YES |
| | 4 | 0.005% | 0.00% | 0.00% | 100 | | YES |

Table 22: Primer specificity results for p190 and p230

Table 23: Primer specificity results for p210(e13a2) and p210(e14a2)

		MR					Acceptable range
Variant	N	Targeted
value	Mean
value	SD	CV, %	Positive
specificity	Positive
specificity	CV,
%	Pass
p210
(e13a2)	4	0.3	0.32	0.003	0.81%	100%	100%