ST AIA-PACK BNP

K192380 · Fujirebio Diagnostics,Inc. · NBC · Aug 24, 2020 · Clinical Chemistry

Device Facts

• Record ID: K192380
• Device Name: ST AIA-PACK BNP
• Applicant: Fujirebio Diagnostics,Inc.
• Product Code: NBC · Clinical Chemistry
• Decision Date: Aug 24, 2020
• Decision: SESE
• Submission Type: Traditional
• Regulation: 21 CFR 862.1117
• Device Class: Class 2

Intended Use

The Tosoh ST AIA-PACK BNP assay is designed for IN VITRO DIAGNOSTIC USE ONLY for the quantitative measurement of BNP in human K2EDTA plasma on Tosoh AIA System analyzers. BNP is used as an aid in the diagnosis of heart failure (HF) in patients presenting to the emergency department (ED) with clinical suspicion of new onset HF, acutely decompensated or exacerbated HF.

Device Story

The ST AIA-PACK BNP is a two-site immunoenzymometric assay performed in test cups on Tosoh AIA System analyzers. It uses magnetic beads coated with anti-BNP mouse monoclonal antibody and enzyme-labeled monoclonal antibody to bind BNP in human K2EDTA plasma. After washing, a fluorogenic substrate (4MUP) is added; the resulting fluorescence is directly proportional to the BNP concentration. The device is used in clinical and hospital laboratories by trained personnel. Results are used by clinicians as an aid in diagnosing heart failure in emergency department patients. The assay provides quantitative BNP measurements, which help clinicians assess heart failure status and severity, potentially improving diagnostic accuracy for patients with suspected cardiac decompensation.

Clinical Evidence

Prospective study of 724 evaluable patients presenting to the ED with suspected heart failure. Performance evaluated against an independent central adjudication panel diagnosis. At a 100 pg/mL cutoff, sensitivity was 88.4% (95% CI: 84.5-91.5%) and specificity was 70.6% (95% CI: 66.0-74.9%). AUC was 0.881. Subgroup analyses by age and gender were performed.

Technological Characteristics

Two-site immunoenzymometric assay. Components: magnetic lyophilized beads coated with anti-BNP mouse monoclonal antibody; anti-BNP mouse monoclonal antibody conjugated to alkaline phosphatase. Energy source: fluorogenic substrate (4-methylumbelliferyl phosphate). Form factor: test cups for use on Tosoh AIA-2000 analyzer. Assay range: 4.0–2000 pg/mL. One-step sandwich assay principle.

Indications for Use

Indicated for patients presenting to the emergency department with clinical suspicion of new onset, acutely decompensated, or exacerbated heart failure.

Regulatory Classification

Identification

The B-type natriuretic peptide (BNP) test system is an in vitro diagnostic device intended to measure BNP in whole blood and plasma. Measurements of BNP are used as an aid in the diagnosis of patients with congestive heart failure.

Special Controls

*Classification.* Class II (special controls). The special control is “Class II Special Control Guidance Document for B-Type Natriuretic Peptide Premarket Notifications; Final Guidance for Industry and FDA Reviewers.”

Predicate Devices

• SIEMENS ADVIA Centaur® BNP Assay (K031038)

Submission Summary (Full Text)

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August 24, 2020

Fujirebio Diagnostics, Inc. Stacey Dolan Sr. Manager, Regulatory Affairs 201 Great Valley Parkway Malvern, PA 19355

Re: K192380

Trade/Device Name: ST AIA-PACK BNP Regulation Number: 21 CFR 862.1117 Regulation Name: B-Type Natriuretic Peptide Test System Regulatory Class: Class II Product Code: NBC Dated: July 14, 2020 Received: July 15, 2020

Dear Stacey Dolan:

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

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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 medical devices and radiation-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 ([e-mail]) or phone [phone] or 301-796-7100).

Sincerely,

Marianela Perez-Torres, Ph.D. Acting Deputy Director Division of Chemistry and Toxicology Devices OHT7: Office of In Vitro Diagnostics and Radiological Health Office of Product Evaluation and Quality Center for Devices and Radiological Health

Enclosure

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# Indications for Use

510(k) Number (if known) k192380

Device Name ST AIA-PACK BNP

Indications for Use (Describe)

The Tosoh ST AIA-PACK BNP assay is designed for IN VITRO DIAGNOSTIC USE ONLY for the quantitative measurement of BNP in human K2EDTA plasma on Tosoh AIA System analyzers. BNP is used as an aid in the diagnosis of heart failure (HF) in patients presenting to the emergency department (ED) with clinical suspicion of new onset HF, acutely decompensated or exacerbated HF.

| Type of Use (Select one or both, as applicable) | |
|--------------------------------------------------------------------------------------|-------------------------------------------------------------------------------------|
| <span style="font-size: 16px;">☑</span> Prescription Use (Part 21 CFR 801 Subpart D) | <span style="font-size: 16px;">☐</span> Over-The-Counter Use (21 CFR 801 Subpart C) |

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### Section 5 510(k) SUMMARY

This summary of 510(k) safety and effectiveness information is being submitted in accordance with the requirements of 21 CFR 807.92.

# A. 510(k) Number:

k192380

# B. Purpose for Submission:

New device

# C. Measurand:

BNP

# D. Type of Test:

Quantitative, Automated Immunoenzymometric assay on the Tosoh AIA System Analyzers

| E. Sponsor: | |
|-----------------|-------------------------------------------------------------------------------------------------------|
| Address: | Tosoh Bioscience, Inc.<br>6000 Shoreline Court, Suite 101<br>South San Francisco, CA 94080 |
| Contact person: | Dave Wurtz<br>Sr. Director Quality, Regulatory and Clinical<br>[phone]<br>[e-mail] |

### F. Application correspondent:

| Address: | Fujirebio Diagnostics, Inc. |
|----------|-----------------------------|
| | 201 Great Valley Parkway |
| | Malvern, PA 19355 |

- Contact person: Stacey Dolan Sr. Manager, Regulatory Affairs [phone] [e-mail]
Summary preparation date: August 20, 2020

# G. Proprietary/Established and Common Names:

- 1. Proprietary/Established Names: ST AIA-PACK BNP
- 2. Common Name: BNP

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Image /page/4/Picture/1 description: The image contains the logo for Fujirebio Diagnostics, Inc. The logo consists of a stylized blue and green graphic on the left, followed by the company name in blue text. The word "FUJIREBIO" is in a larger, bolder font than "Diagnostics, Inc."

### H. Regulatory Information:

- 1. Requlation section: 21 CFR § 862.1117, Test, Natriuretic Peptide
- 2. Classification: Class II
- 3. Product code: NBC, B-Type natriuretic peptide test system
- 4. Panel: 75, Chemistry
- l. Intended Use:
- 1. Intended use(s): See indications for use below.
- 2. Indication(s) for use:

The Tosoh ST AIA PACK BNP assay is designed for IN VITRO DIAGNOSTIC USE ONLY for the quantitative measurement of BNP in human (K2EDTA) plasma on Tosoh AIA System Analyzers. BNP is used as an aid in the diagnosis of heart failure (HF) in patients presenting to the emergency department (ED) with clinical suspicion of new onset HF, acutely decompensated or exacerbated HF.

- 3. Special conditions for use statement(s): Prescription use only
- 4. Special instrument requirements: Tosoh AIA System Analyzers

# J. Device Description:

The ST AIA-PACK BNP is a two-site immunoenzymometric assay which is performed entirely in the ST AIA-PACK BNP test cups. BNP present in the test sample is bound with monoclonal antibody immobilized on magnetic beads and enzyme-labeled monoclonal antibody. The magnetic beads are washed to remove unbound enzyme-labeled monoclonal antibody and are then incubated with a fluorogenic substrate, 4-methylumbelliferyl phosphate (4MUP). The amount of enzyme-labeled monoclonal antibody that binds to the beads is directly proportional to the BNP concentration in the test sample. A standard curve is constructed, and unknown sample concentrations are calculated using the curve.

### ST AIA-PACK BNP (Cat. No. 025228)

The ST AIA-PACK BNP set consists of 5 trays x 20 test cups. Each kit contains plastic test cups containing twelve magnetic lyophilized beads coated with anti-BNP mouse monoclonal antibody and 100 µL of anti-BNP mouse monoclonal antibody conjugated to alkaline phosphatase with sodium azide as a preservative.

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### Other Materials/Equipment Required (not Provided):

### AIA Systems: AIA-2000 ST AIA-2000 LA

# AIA PACK

AIA-PACK Substrate Set II AIA-PACK Substrate Reagent II/ AIA-PACK Substrate Reconstituent II AIA-PACK Wash Concentrate AIA-PACK Diluent Concentrate Sample Cups AIA-PACK Detector Standardization Test Cups Pipette Tips (1000/pkg) Tip Rack (empty) Preloaded Pipette Tips (96 tips x 50 racks) Preloaded Pipette Tips (96 Tips x 5 Racks)

# K. Substantial Equivalence Information:

- 1. Predicate device name(s): SIEMENS ADVIA Centaur® BNP Assay
- 2. Predicate 510(k) number(s): K031038
- 3. Comparison with predicate:

| Similarities | | |
|----------------|-----------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------|------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------|
| | ST AIA-PACK BNP<br>(Proposed Device) | SIEMENS ADVIA Centaur® BNP<br>(Predicate Device)<br>K031038 |
| Device Type | In vitro diagnostic | In vitro diagnostic |
| Classification | Class II | Class II |
| CFR section | 21 CFR 862.1117 | 21 CFR 862.1117 |
| Product Code | NBC | NBC |
| Product Usage | Clinical and Hospital laboratories | Clinical and Hospital laboratories |
| Intended Use | The Tosoh ST AIA PACK BNP<br>assay is designed for IN VITRO<br>DIAGNOSTIC USE ONLY for the<br>quantitative measurement of BNP<br>in human (K₂EDTA) plasma on<br>Tosoh AIA System Analyzers. BNP<br>is used as an aid in the diagnosis of<br>heart failure in patients presenting | For in vitro diagnostic use in the<br>quantitative determination of B-<br>type Natriuretic Peptide (BNP) in<br>human plasma using the ADVIA<br>Centaur and ADVIA Centaur XP<br>systems. This assay is indicated<br>for the measurement of plasma<br>BNP as an aid in the diagnosis |

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Image /page/6/Picture/1 description: The image is a logo for Fujirebio Diagnostics, Inc. The logo features a stylized graphic on the left, consisting of blue and green shapes. To the right of the graphic is the company name, "FUJIREBIO", in large, bold, blue letters. Below the company name is the text "Diagnostics, Inc.", in a smaller font size.

| Similarities | | |
|-------------------------------|-----------------------------------------------------------------------------------|-------------------------------------------------------------|
| | ST AIA-PACK BNP<br>(Proposed Device) | SIEMENS ADVIA Centaur® BNP<br>(Predicate Device)<br>K031038 |
| | to the emergency department (ED)<br>with symptoms suggestive of heart<br>failure. | and assessment of the severity of<br>heart failure. |
| Type of Specimen | Human K₂EDTA plasma | Human EDTA Plasma |
| Specimen Collection<br>Method | Routine Phlebotomy Techniques | Routine Phlebotomy Techniques |
| Analyte | Human B-type Natriuretic Peptide<br>(BNP) | Human B-type Natriuretic Peptide<br>(BNP) |
| Cut-off | 100 pg/mL | 100 pg/mL |

| Differences | | |
|------------------------|-------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------|------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------|
| | ST AIA-PACK BNP<br>(Proposed Device) | SIEMENS ADVIA Centaur® BNP<br>(Predicate Device)<br>K031038 |
| Instrument System | Tosoh AIA Analyzer 2000 | ADVIA Centaur and ADVIA<br>Centaur XP Systems |
| Principle of Operation | Immunoenzymometric Assay | Chemiluminescence immunoassay |
| Assay Range | 4.0 - 2000pg/mL | <2.0 – 5000pg/mL |
| Detection | Fluorescence | Chemiluminescence |
| Labeled antibody | Alkaline phosphatase labeled<br>mouse monoclonal (mAb KY-BNP-<br>II) anti-human BNP (Fab')2<br>fragment specific to the ring<br>structure of BNP. Labeling was<br>done by introducing a malemide<br>group at the hinge portion of the<br>Fab'2 fragment and reacting it with<br>alkaline phosphatase. | Acridinium ester labeled mouse<br>monoclonal (mAb KY-BNP-II) anti-<br>human BNP (Fab')2 fragment<br>specific to the ring structure of<br>BNP. |
| Solid phase antibody | Mouse monoclonal anti-human<br>BNP (intact) antibody (mAb BC-<br>203) specific to the C-terminal<br>portion of BNP, which is<br>immobilized on magnetic beads | Biotinylated monoclonal mouse<br>anti-human antibody (mAb BC-<br>203) specific to the C-terminal<br>portion of BNP which is coupled to<br>streptavidin magnetic particles |
| Test principle | One-step sandwich assay | Delayed one-step sandwich assay |
| Incubation time | ~ 10 minutes at 37° C | First - 5 minutes at 37° C<br>Second - 2.5 minutes at 37° C |
| Reaction | Unbound enzyme-labeled<br>monoclonal antibody is washed<br>away and a fluorogenic substrate,<br>4-methylumbelliferyl phosphate<br>(4MUP) is added. The degree of<br>fluorescence is directly | Following the second incubation,<br>the unbound antibody conjugates<br>are washed away. An immune-<br>complex is formed between the<br>BNP in the sample and the two<br>antibody conjugates. The amount<br>of relative light units detected by |

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Image /page/7/Picture/1 description: The image contains the logo for Fujirebio Diagnostics, Inc. The logo features a stylized graphic on the left, consisting of blue and green shapes that resemble a stylized water droplet or leaf. To the right of the graphic, the text "FUJIREBIO" is written in a bold, blue sans-serif font. Below "FUJIREBIO", the words "Diagnostics, Inc." are written in a smaller, black sans-serif font.

| proportional to the amount of BNP in the sample. | the system is directly proportional to the amount of BNP in the sample. |
|--------------------------------------------------|-------------------------------------------------------------------------|
|--------------------------------------------------|-------------------------------------------------------------------------|

# L. Standard/Guidance Document Referenced (if applicable):

- . CLSI C28-A3c: Defining, Establishing and Verifying Reference Intervals in the Clinical Laboratory; Approved Guideline
- . CLSI EP06-A: Evaluation of Linearity of Quantitative Measurement Procedures, A Statistical Approach: Approved Guideline
- CLSI EP7-A2: Interference Testing in Clinical Chemistry; Approved Guideline
- CLSI EP07: Interference Testing in Clinical Chemistry; Approved Guideline – Third Edition
- CLSI EP37: Supplemental Tables for Interference Testing in Clinical Chemistry: Approved . Guideline - First Edition
- CLSI EP17-A: Protocols for Determination of Limits of Detection and Limits of Quantitation: Approved Guideline
- . CLSI EP5-A2: Evaluation of Precision Performance of Quantitative Measurement Methods; Approved Guideline
- CLSI EP05-A3: Evaluation of Precision of Quantitative Measurement Procedures; Approved Guideline - Third Edition
- . Guidance on Informed Consent for In Vitro Diagnostic Device Studies Using Leftover Human Specimens that are Not Individually Identifiable; Guidance for Sponsors, Institutional Review Boards, Clinical Investigators, and Food and Drug Administration Staff (April 25, 2006)
- . Class II Special Control Guidance Document for B-Type Natriuretic Peptide Premarket Notifications; Final Guidance for Industry and FDA Reviewers. Document issued on: November 30, 2000

# M. Test Principle:

The ST AIA-PACK BNP is a two-site immunoenzymometric assay which is performed entirely in the ST AIA-PACK BNP test cups. BNP present in the test sample is bound with monoclonal antibody immobilized on magnetic beads and enzyme-labeled monoclonal antibody. The magnetic beads are washed to remove unbound enzyme-labeled monoclonal antibody and are then incubated with a fluorogenic substrate, 4-methyllumbellifery| phosphate (4MUP). The amount of enzyme-labeled monoclonal antibody that binds to the beads is directly proportional to the BNP concentration in the test sample. A standard curve is constructed, and unknown sample concentrations are calculated using this curve.

# N. Performance Characteristics (if/when applicable):

- 1. Analytical performance:
- Precision/Reproducibility: a.

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Image /page/8/Picture/1 description: The image shows the logo for Fujirebio Diagnostics, Inc. The logo consists of a stylized, abstract symbol on the left, followed by the company name on the right. The symbol is composed of three curved shapes, two in solid blue and one with vertical green lines, arranged to create a sense of unity. The text "FUJIREBIO" is in bold, blue letters, and below it, "Diagnostics, Inc." is written in a smaller, thinner font.

CLSI Guideline EP5-A2 entitled: "Evaluation of Precision Performance of Quantitative measurement Methods; Approved Guideline - Second Edition. was used to design and CLSI Guideline EP05-A3 entitled: Evaluation of Quantitative Measurement Procedures; Approved Guideline - Third Edition was used to analyze the within run, between run, between day, between lot and total precision studies. The study was conducted at 1 site using three (3) different lots of ST AIA-PACK BNP Reagents and Calibrator Sets on three (3) different AIA-2000 analyzers to assess the precision of the ST AIA-PACK BNP assay.

Six (6) K2EDTA plasma samples were tested. The compositions and concentrations are described below in the table below.

| Sample Number | Approximate<br>Concentration (pg/mL) | Sample Composition |
|-----------------|--------------------------------------|------------------------------------------------------------------------------------------------------|
| K2EDTA Plasma-1 | 10 | Apparently healthy<br>patient plasma sample |
| K2EDTA Plasma-2 | 50 | Apparently healthy<br>patient plasma sample +<br>patient plasma sample<br>expressing elevated<br>BNP |
| K2EDTA Plasma-3 | 100 | Apparently healthy<br>patient plasma sample +<br>patient plasma sample<br>expressing elevated<br>BNP |
| K2EDTA Plasma-4 | 500 | Apparently healthy<br>patient plasma sample<br>augmented with BNP<br>antigen |
| K2EDTA Plasma-5 | 1000 | Apparently healthy<br>patient plasma sample<br>augmented with BNP<br>antigen |

Within run, between run, between day and total precision was calculated for each lot combination of ST AIA-PACK BNP Reagents and Calibrator Sets and are summarized in the tables below. In addition, precision was calculated for the combined lots as summarized in the following tables.

ST AIA-PACK BNP Precision Lot 1(n=80)

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| Sample | | K2EDTA<br>Plasma-1 | K2EDTA<br>Plasma-2 | K2EDTA<br>Plasma-3 | K2EDTA<br>Plasma-4 | K2EDTA<br>Plasma-5 |
|-----------------------|-----|--------------------|--------------------|--------------------|--------------------|--------------------|
| Mean Conc.<br>(pg/mL) | | 11.383 | 50.676 | 108.286 | 518.967 | 1059.189 |
| Within Run | SD | 0.529 | 1.534 | 2.449 | 9.763 | 20.342 |
| | %CV | 4.6 | 3.0 | 2.3 | 1.9 | 1.9 |
| Between<br>Run | SD | 0.000 | 0.000 | 0.000 | 2.304 | 12.679 |
| | %CV | 0.0 | 0.0 | 0.0 | 0.4 | 1.2 |
| Between<br>Day | SD | 0.000 | 0.000 | 0.000 | 0.000 | 4.452 |
| | %CV | 0.0 | 0.0 | 0.0 | 0.0 | 0.4 |
| Total | SD | 0.529 | 1.534 | 2.449 | 10.031 | 24.380 |
| | %CV | 4.6 | 3.0 | 2.3 | 1.9 | 2.3 |

### ST AIA-PACK BNP Precision Lot 2 (n=80)

| Sample | K2EDTA<br>Plasma-1 | K2EDTA<br>Plasma-2 | K2EDTA<br>Plasma-3 | K2EDTA<br>Plasma-4 | K2EDTA<br>Plasma-5 | |
|--------------------|--------------------|--------------------|--------------------|--------------------|--------------------|--------|
| Mean Conc. (pg/mL) | 10.896 | 49.919 | 104.864 | 495.956 | 988.208 | |
| Within Run | SD | 0.609 | 1.450 | 1.997 | 11.611 | 25.244 |
| | %CV | 5.6 | 2.9 | 1.9 | 2.3 | 2.6 |
| Between Run | SD | 0.000 | 0.307 | 1.777 | 7.232 | 15.589 |
| | %CV | 0.0 | 0.6 | 1.7 | 1.5 | 1.6 |
| Between Day | SD | 0.361 | 0.722 | 0.541 | 0.000 | 5.415 |
| | %CV | 3.3 | 1.4 | 0.5 | 0.0 | 0.5 |
| Total | SD | 0.708 | 1.648 | 2.728 | 13.679 | 30.159 |
| | %CV | 6.5 | 3.3 | 2.6 | 2.8 | 3.1 |

ST AIA-PACK BNP Precision Lot 3 (n=80)

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Image /page/10/Picture/1 description: The image shows the logo for Fujirebio Diagnostics, Inc. The logo consists of a blue and green abstract shape on the left, followed by the company name in blue, with "Diagnostics, Inc." in a smaller font size below. The logo is clean and professional, with a focus on the company's name and brand.

| Sample | | K2EDTA<br>Plasma-1 | K2EDTA<br>Plasma-2 | K2EDTA<br>Plasma-3 | K2EDTA<br>Plasma-4 | K2EDTA<br>Plasma-5 |
|--------------------|-----|--------------------|--------------------|--------------------|--------------------|--------------------|
| Mean Conc. (pg/mL) | | 9.486 | 49.023 | 107.004 | 543.364 | 1104.740 |
| Within Run | SD | 0.494 | 1.642 | 3.016 | 8.806 | 18.148 |
| | %CV | 5.2 | 3.3 | 2.8 | 1.6 | 1.6 |
| Between<br>Run | SD | 0.000 | 0.000 | 0.000 | 6.076 | 12.407 |
| | %CV | 0.0 | 0.0 | 0.0 | 1.1 | 1.1 |
| Between Day | SD | 0.120 | 0.000 | 0.000 | 0.000 | 0.000 |
| | %CV | 1.3 | 0.0 | 0.0 | 0.0 | 0.0 |
| Total | SD | 0.508 | 1.642 | 3.016 | 10.699 | 21.984 |
| | %CV | 5.4 | 3.3 | 2.8 | 2.0 | 2.0 |

# ST AIA-PACK BNP Precision Combined Lots (n=240)

| ST AIA-PACK BNP Precision Combined Lots (n=240) | | | | |
|-------------------------------------------------|-------------------------|---------------------|---------------------------|------|
| Sample ID | Overall Mean<br>(pg/mL) | Source of Variation | Pooled Standard Deviation | %CV |
| K2EDTA<br>Plasma-1 | 10.588 | Within Run | 0.547 | 5.2 |
| | | Between Run | 0.000 | 0.0 |
| | | Between Day | 0.217 | 2.0 |
| | | Between Lot | 0.982 | 9.3 |
| | | Total | 1.145 | 10.8 |
| K2EDTA<br>Plasma-2 | 49.873 | Within Run | 1.592 | 3.2 |
| | | Between Run | 0.000 | 0.0 |
| | | Between Day | 0.219 | 0.4 |
| | | Between Lot | 0.807 | 1.6 |
| | | Total | 1.798 | 3.6 |
| K2EDTA<br>Plasma-3 | 106.718 | Within Run | 2.637 | 2.5 |
| | | Between Run | 0.739 | 0.7 |
| | | Between Day | 0.000 | 0.0 |
| | | Between Lot | 1.700 | 1.6 |
| | | Total | 3.223 | 3.0 |
| K2EDTA<br>Plasma-4 | 519.429 | Within Run | 10.127 | 1.9 |
| | | Between Run | 5.613 | 1.1 |
| | | Between Day | 0.000 | 0.0 |
| | | Between Lot | 23.664 | 4.6 |
| | | Total | 26.346 | 5.1 |
| K2EDTA<br>Plasma-5 | 1050.712 | Within Run | 21.451 | 2.0 |
| | | Between Run | 13.823 | 1.3 |
| | | Between Day | 3.329 | 0.3 |
| | | Between Lot | 58.632 | 5.6 |
| | | Total | 64.031 | 6.1 |

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Image /page/11/Picture/1 description: The image shows the logo for Fujirebio Diagnostics, Inc. The logo consists of a blue and green abstract symbol on the left, followed by the company name in blue text. The word "FUJIREBIO" is in a larger, bolder font than "Diagnostics, Inc.", which is placed below it.

#### b. Linearity/assay reportable range:

The CLSI Guideline EP6-A entitled: "Evaluation of the Linearity of Quantitative Measurement Procedures; A Statistical Approach" Approved Guideline was used to design the linearity study. A total of fourteen (14) samples ranging from 3.1 - 2271.5 pg/mL were assayed to determine linearity. The assay has been demonstrated to be linear from 4.0 to 2000 pg/mL.

| Dilution | | Expected<br>Value | Mean | SD | CV<br>(%) | Recovery<br>(%) |
|----------|-------------------|-------------------|---------|-------|-----------|-----------------|
| No. | Low : High | (pg/mL) | (pg/mL) | | | |
| | Low<br>Undiluted | | | | | |
| 1 | Undiluted | 3.10 | 3.28 | 1.19 | 36.2 | 105.6 |
| 1.18 | 9.85 : 0.15 | 35.04 | 34.61 | 1.11 | 3.2 | 98.8 |
| 1.3 | 9.7 : 0.3 | 68.50 | 67.83 | 1.07 | 1.6 | 99.0 |
| 1.6 | 9.4: 0.6 | 135.88 | 137.35 | 1.01 | 0.7 | 101.1 |
| 2 | 9 : 1 | 223.19 | 226.19 | 3.28 | 1.4 | 101.3 |
| 3 | 8 : 2 | 453.41 | 459.57 | 4.31 | 0.9 | 101.4 |
| 4 | 7 : 3 | 686.42 | 688.68 | 12.98 | 1.9 | 100.3 |
| 5 | 6 : 4 | 905.01 | 915.36 | 11.64 | 1.3 | 101.1 |
| 6 | 5 : 5 | 1139.99 | 1164.54 | 23.01 | 2.0 | 102.2 |
| 7 | 4 : 6 | 1361.66 | 1387.54 | 10.51 | 0.8 | 101.9 |
| 8 | 3 : 7 | 1584.01 | 1619.32 | 7.13 | 0.4 | 102.2 |
| 9 | 2 : 8 | 1821.03 | 1842.89 | 18.47 | 1.0 | 101.2 |
| 10 | 1 : 9 | 2044.07 | 2072.97 | 33.01 | 1.6 | 101.4 |
| | High<br>Undiluted | | | | | |
| 11 | Undiluted | 2271.54 | 2289.36 | 20.47 | 0.9 | 100.8 |

#### C. Traceability, Stability, Expected values (controls, calibrators, or methods):

### Traceability:

There is currently no known internationally recognized consensus reference method or reference material for standardization. BNP assay values are expressed as pg/mL. The ST AIA-PACK BNP CALIBRATOR SET contains assigned concentrations of BNP. The assigned value is determined on a lot-by-lot basis and is designed to provide an assay calibration range of 4.0 to 2,000 pg/mL of BNP. The calibrators in this set are prepared gravimetrically and are compared to internal reference standards.

### Stability:

The stability data supports the current shelf life assignment for the ST AIA-PACK BNP of 12 months.

### Expected Values:

- d. Detection limit:

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The CLSI Guideline EP17-A entitled: Protocols for Determination of Limits of Detection and Limits of Quantitation; Approved Guideline was used to design the LoD and LoQ study.

Eleven low level samples selected from concentrations in the range from 1.341 to 17.009 pg/mL (LoB to 4x LoB) were prepared by dilution of specimens with known BNP concentrations. The samples were assayed in replicates of two (2) over five (5) days on one instrument for a total of ten (10) replicates per sample. The standard deviation (SD) and coefficient of variation (CV%) were calculated

LoQ was calculated as the functional sensitivity at 20% CV. To determine the functional sensitivity, a precision profile was plotted using the values of CV% and the mean concentration of the samples in pq/mL from the LoD study.

LoB = 0.9 pg/mL LoD = 1.9 pg/mL LoQ = 3.5 pg/mL.

- Analytical specificity: e.
Studies were conducted to evaluate the potential interference from the endogenous substance summarized below with the Tosoh ST AIA-PACK BNP assay. CLSI Guideline, EP7-A2 entitled: Interference Testing in Clinical Chemistry - Approved Guideline, CLSI Guideline EP07 entitled Interference Testing in Clinical Chemistry; Approved Guideline - Third Edition and CLSI Guideline EP37 entitled Supplemental Tables for Interference Testing in Clinical Chemistry: Approved Guideline - First Edition were used to design the interference study. KչEDTA samples with known concentrations of BNP, at approximately 35 pg/mL, 90 pg/mL and 1,000 pg/mL were spiked with varying concentrations of the potential interferents.

The criterion for no interference is +/- 10% recovery of BNP in the known specimen mean concentration.

Hemoglobin up to 130 mg/dL does not interfere with the assay.

Unconjugated (Free) Bilirubin up to 15 mg/dL does not interfere with the assay.

Conjugated Bilirubin up to 19 mg/dL does not interfere with the assay.

Lipemia (represented by triglycerides) up to 1600 mg/dL does not interfere with the assay.

Total Protein (represented by human serum albumin) up to 14 g/dL does not interfere with the assay.

Ascorbic Acid up to 20 mg/dL does not interfere with the assay.

Rheumatoid Factor up to 500 IU/mL does not interfere with the assay.

Human IgG up to 5.3 g/dL does not interfere with the assay.

Cholesterol up to 400 mg/dL does not interfere with the assay.

Creatinine up to 15 mg/dL does not interfere with the assay.

Alkaline Phosphatase up to 2000 U/L does not interfere with the assay.

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HAMA IgG up to 500 ng/mL does not interfere with the assay.

Studies were conducted to evaluate the potential interference from various compounds with the Tosoh ST AI-PACK BNP assay. K2EDTA plasma samples with measurable amounts of BNP were spiked with each cross reactant at a concentration of 50, 600 or 1000 pg/mL. Results are summarized below.

| Cross Reactant | Test Level<br>(pg/mL) | % Cross<br>Reactivity |
|-------------------------|-----------------------|-----------------------|
| Adrenomedullin 52 Human | 1000 | 0.17 |
| Aldosterone | 1000 | 0.11 |
| Angiotensin I | 600 | -0.02 |
| Angiotensin II | 600 | -0.13 |
| Angiotensin III | 1000 | -0.37 |
| ANP | 1000 | 0.03 |
| Arg8-Vasopressin | 1000 | 0.02 |
| CNP | 1000 | 0.00 |
| DNP | 1000 | 0.43 |
| Endothelin | 1000 | -0.08 |
| NT-proBNP | 1000 | -0.05 |
| Renin | 50 | 0.18 |
| Urodilatin | 1000 | 0.00 |
| VNP | 900 | 1.63 |

Studies were conducted to evaluate the potential interference from several therapeutics with the Tosoh ST AIA-PACK BNP assay. K2EDTA samples of known BNP concentrations, targeting 100 pg/mL and 300 – 500 pg/mL were used for the study. The therapeutic agent was spiked into each specimen, and the dilution factor was <5% for each agent. The compound and the observed interference for each therapeutic agent are listed below. The % recovery for each therapeutic interferent was within 100+10% of the control.

| Compound Name | Test Level | Low Level<br>Target<br>100 pg/ml<br>% recovery | High Level<br>Target<br>300-500 pg/ml<br>% recovery |
|--------------------------------------------------------------|-----------------|------------------------------------------------|-----------------------------------------------------|
| Acetaminophen (4-<br>Acetamidophenol) | 220 µg/mL | 96 | 99 |
| Acetylsalicylic Acid | 200 µg/mL | 100 | 97 |
| Allopurinol | 240 ug/mL | 98 | 101 |
| Amiodarone | 4.2 mg/dL | 107 | 97 |
| Amlodipine besylate | 4 µg/mL | 97 | 98 |
| Ampicillin | 200 µg/mL | 100 | 102 |
| L-Ascorbic Acid | 66.2 µg/mL | 104 | 102 |
| Atenolol | 40 µg/mL | 100 | 101 |
| Atorvastatin | 32 µg/mL | 98 | 105 |
| Biotin | 30 µg/mL | 100 | 101 |
| Caffeine | 10.8 mg/dL | 99 | 100 |
| Carvedilol | 30 µg/mL | 103 | 104 |
| Compound Name | Test Level | Low Level<br>Target<br>100 pg/ml<br>% recovery | High Level<br>Target<br>300-500 pg/ml<br>% recovery |
| Captopril | 40 µg/mL | 97 | 95 |
| Chloramphenicol | 7.8 mg/dL | 107 | 100 |
| Clopidogrel Bisulfate | 30 µg/mL | 101 | 98 |
| Cyclosporine | 40 µg/mL | 99 | 100 |
| Diclofenac sodium salt | 60 µg/mL | 103 | 98 |
| Digitoxin | 60 µg/mL | 97 | 98 |
| Digoxin | 0.0039<br>mg/dL | 100 | 97 |
| (+)-cis-Diltiazem hydrochloride | 120 µg/mL | 97 | 101 |
| Dipyridamole | 30 µg/mL | 96 | 100 |
| Disopyramide | 1.68 mg/dL | 101 | 103 |
| Dobutamine | 100 µg/mL | 99 | 99 |
| Dopamine hydrochloride | 116 µg/mL | 103 | 98 |
| Enalaprilat dehydrate (hydrolyzed<br>from enalapril maleate) | 16 µg/mL | 100 | 103 |
| Erythromycin | 13.8 mg/dL | 107 | 97 |
| Fenofibrate | 45 µg/mL | 99 | 101 |
| Furosemide | 65.9 µg/mL | 94 | 92 |
| Heparin | 330 units/dL | 101 | 99 |
| Hydralazine | 20 µg/mL | 100 | 100 |
| Hydrochlorothiazide | 20 µg/mL | 98 | 99 |
| Ibuprofen | 500 µg/mL | 106 | 103 |
| Indomethacin | 36 µg/mL | 104 | 100 |
| Isosorbide dinitrate | 0.593 mg/dL | 101 | 100 |
| Levothyroxine | 0.0429<br>mg/dL | 91 | 94 |
| Lidocaine | 1.5 mg/dL | 100 | 97 |
| Lisinopril x 2H2O | 16 µg/mL | 96 | 99 |
| Losartan potassium | 59.9 µg/mL | 99 | 101 |
| Lovastatin | 0.021 mg/dL | 103 | 102 |
| Methyldopa | 100 µg/mL | 103 | 101 |
| (±)-Metoprolol (+)-tartrate salt | 12.8 µg/mL | 102 | 100 |
| Naproxen | 499 µg/mL | 101 | 102 |
| Nicotine | 1.6 µg/mL | 102 | 100 |
| Nicotinic acid | 40 µg/mL | 99 | 98 |
| Nifedipine | 36 µg/mL | 98 | 100 |
| Nitrofuratoin | 40 µg/mL | 101 | 102 |
| Oxazepam | 12 µg/mL | 100 | 98 |
| Oxytetracycline | 100 µg/mL | 99 | 102 |
| Phenobarbital | 69 mg/dL | 99 | 103 |
| Phenytoin | 6.00 mg/dL | 96 | 103 |
| Compound Name | Test Level | Low Level<br>Target<br>100 pg/ml<br>% recovery | High Level<br>Target<br>300-500 pg/ml<br>% recovery |
| Probenecid | 600 µg/mL | 100 | 98 |
| Procainamide | 4.80 mg/dL | 96 | 109 |
| Propanolol | 64 µg/mL | 102 | 102 |
| Quinidine | 20 µg/mL | 99 | 102 |
| Ramipril | 6 µg/mL | 101 | 102 |
| Simvastatin | 32 µg/mL | 99 | 102 |
| Spironolactone | 600 µg/mL | 101 | 100 |
| Sulfamethoxazole | 0.43 µg/mL | 95 | 103 |
| Theophylline | 6.00 mg/dL | 101 | 101 |
| Trymethoprim | 64 µg/mL | 99 | 100 |
| Verapamil hydrochloride | 96 µg/mL | 100 | 105 |
| Warfarin | 7.5 mg/dL | 109 | 100 |
| Trasylol/Aprotinin | 100 KIE/mL | 97 | 100 |

Tosoh ST AIA-Pack BNP Therapeutic Interference Results

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#### f. Assay cut-off:

See Clinical Cutoff in M (5) below

- Comparison studies: 2. N/A
#### 3. Clinical studies:

This study was conducted to determine the performance of the ST AIA-PACK BNP assay at the current cutoff of BNP at 100 pg/mL as recommended in the American Heart Association guidelines for acute heart failure (HF). Additional analysis was conducted to determine performance of the assay by gender and age. Performance was analyzed separately among patients with and without comorbidities. Statistics were performed between these sub-groups to determine if there were any significant differences in performance.

The prospective study enrolled male and female patients from 8 clinical sites comprised of Emergency Departments (ED). The study included patients who were presented to the ED with clinical suspicion of new onset HF, acutely decompensated or exacerbated HF, where a clinician would order a BNP test as part of a differential diagnosis. Patient samples were stored at -20°C or colder then sent to a central location for testing at a Tosoh Bioscience lab. A total of 825 samples were assayed for BNP using the Tosoh AIA 2000 Analyzer. Out of the 825 samples, a total number of 724 samples were used for the analysis. 101 samples were excluded from the analysis. Most of these exclusions were due to hemolysis and patients with severe renal insufficiency requiring dialysis (estimated glomerular filtration rate (eGFR) < 30 mL/min/1.73m2): Chronic Kidney Disease (CKD) Stage 4 and 5.

The participants in this study were categorized into the following races: White (n=483, 66.7%), Black (n=144, 19.9%) and other (n=97, 13.3%). The population tested was fairly balanced with respect to females (n=327) versus males (n=397) and older (age ≥ 75 years, n=247) versus younger (age < 75 years, n= 477) subjects. Below is the Age Demographics for Evaluable Subjects.

### Summary of Age Demographics for Evaluable Subjects

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| Age Group<br>(years) | All |
|----------------------------|------|
| 22-29 | 11 |
| 30-39 | 28 |
| 40-49 | 55 |
| 50-59 | 140 |
| 60-69 | 159 |
| 70-79 | 155 |
| 80-89 | 136 |
| ≥ 90 | 40 |
| Total | 724 |
| Mean Age | 67 |
| Median Age | 67 |
| Standard Deviation<br>(SD) | 15.3 |
| Minimum Age | 24 |
| Maximum Age | 97 |

Diagnosis of HF or non- HF was determined by an independent central adjudication panel in order to ensure standardization and accuracy of diagnosis per 2013 ACCF/AHA Guidelines for Management of HF.

The adjudication panel (comprised of 4 expert cardiologists and 1 ER physician) had access to patient CRFs and clinical information, including but not limited to, echocardiography, and other cardiac and thoracic imaging, and standard of care BNP or B-type Natriuretic Peptide, N-terminal Pro B-type Natriuretic Peptide (NT-proBNP) results, if available.

The adjudication panel was blinded to the attending physician's final diagnosis and NYHA classification.

# Patients with Heart Failure (HF)

Among the 724 subjects who presented to the ED, 329 patients were determined to have HF by the adjudication panel. All of the patients with HF in the study were categorized utilizing the New York Heart Association (NYHA) Classification system. The NYHA is a four-stage assessment tool that classifies the stage of heart failure based on the subjective observation of a patient's clinical signs and symptoms. It is based on the patient's limitations in physical activity, difficulty with regard to breathing, and angina pain. Below is a description of the classification tool.

### NYHA Classification - The Stages of Heart Failure:

| NYHA Classification | |
|---------------------|--|
| | |

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| Class I | No symptoms and no limitation in ordinary physical activity |
|-----------|-----------------------------------------------------------------------------------------|
| Class II | Mild shortness of breath and/or angina and slight limitation during ordinary activity |
| Class III | Marked limitation in activity due to symptoms, even during less- than ordinary activity |
| Class IV | Severe limitations. Experiences symptoms even while at rest |

The tables below reflect the BNP levels for all HF patients stratified by their NYHA classification.

### Heart Failure Population: All

| | All HF | NYHA I | NYHA II | NYHA III | NYHA IV |
|------------------|--------|--------|---------|----------|---------|
| Sample size (N=) | 329 | 2 | 51 | 177 | 99 |
| Mean(pg/mL) | | 119.0 | 323.1 | 673.4 | 565.9 |
| SD (pg/mL) | | 69.6 | 353.4 | 1074.6 | 518.8 |
| Median (pg/mL) | | 119.0 | 214.7 | 449.0 | 420.6 |
| 95th percentile | | 163.0 | 1124.0 | 1814.0 | 1418.0 |

The BNP cutoff of 100 pg/mL is recommended in the American Heart Association guidelines for acute heart failure (HF). BNP level increase is positively correlated with the severity of heart failure.

The cross tabulation of results between HF and BNP at the cutoff of 100 pg/mL is below.

### Cross-tabulation of Patients by Current cutoff: ST AIA PACK BNP Assay

| | BNP | | |
|-------------|------------|------------|-------|
| Adjudicated | ≥100 pg/mL | <100 pg/mL | Total |
| HF | 291 | 38 | 329 |
| Not HF | 116 | 279 | 395 |
| Total | 407 | 317 | 724 |

Using the traditional single cutoff of 100 pg/mL, the sensitivity of the ST AIA-PACK BNP assay is 88.4% and the specificity is 70.6%. The Positive Value (PPV) is 71.5% and the Negative Predictive Value (NPV) is 88.0%. The prevalence of HF was 45.4% and of no HF was 54.6%

### Overall Performance of ST AIA-PACK BNP Assay

| Measure | Value | Low CI* | High CI* |
|---------|-------|---------|----------|
|---------|-------|---------|----------|

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| Sensitivity | 88.4% | 84.5% | 91.5% |
|----------------------------------------|--------|--------|--------|
| Specificity | 70.6% | 66.0% | 74.9% |
| Sensitivity +<br>Specificity | 159.1% | 153.0% | 164.3% |
| Concordance | 78.7% | 75.6% | 81.6% |
| PPV | 71.5% | 66.9% | 75.7% |
| NPV | 88.0% | 84.0% | 91.1% |
| Prevalence of HF | 45.4% | 41.8% | 49.1% |
| Prevalence of no HF | 54.6% | 50.9% | 58.2% |
| Positive Likelihood<br>Ratio (PLR), CI | 3.012 | 2.572 | 3.527 |
| Negative Likelihood<br>Ratio (NLR), CI | 0.164 | 0.120 | 0.222 |

*95% CI determined using Wilson Score

Note: subgroup analyses can be found below and may be different than the overall performance.

### Receiver Operator Characteristic (ROC)

The utility of BNP as a diagnostic marker for HF is described in multiple reports in the scientific literature. Data from the clinical study was used to generate the Receiver Operating Characteristic (ROC) curve of BNP decision thresholds versus the clinical sensitivity and clinical specificity as shown in Figure 1. The area under the curve (AUC) is 0.881.

Image /page/18/Figure/7 description: This image shows a plot of sensitivity versus false positives. The plot shows a curve that starts at the bottom left corner and rises to the top right corner. The area under the curve (AUC) is 0.881, and the standard error (SE) is 0.013. There is a vertical line at cutoff 100.

# ROC curve for BNP

Figure 1. Receiver Operator Curve

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#### 4. Clinical cut-off

See M (5) below

#### 5. Expected values/Reference Range

CLSI Guideline C28-A3 entitled: "Defining, Establishing, and Verifying Reference Intervals in the Clinical Laboratory, Approved Guideline" – Third Edition was used to design the reference range study. All specimens were assayed in single replicates utilizing the ST AIA-PACK BNP assay.

To establish the reference range, K2EDTA plasma samples obtained from apparently healthy males and females were tested using the ST AIA-PACK BNP. The descriptive statistics for BNP concentrations in the apparently healthy (normal) population is shown in the table below.

| Reference Group: All | | | | | | |
|----------------------|-------|-------|-------|-------|-------|-------|
| | Age | | | | | |
| | All | <45 | 45-54 | 55-64 | 65-74 | ≥75 |
| N | 430 | 92 | 83 | 88 | 87 | 80 |
| Mean pg/mL | 34.6 | 10.4 | 11.9 | 15.1 | 58.1 | 81.6 |
| SD pg/mL | 73.8 | 11.0 | 11.2 | 18.1 | 106.6 | 110.2 |
| Median pg/mL | 10.8 | 5.9 | 7.9 | 8.5 | 14.7 | 39.1 |
| 95 percentile pg/mL | 143.2 | 26.0 | 39.8 | 42.5 | 241.2 | 273.6 |
| % < 100 pg/mL | 91.4 | 100.0 | 100.0 | 98.9 | 82.8 | 73.8 |
| Minimum pg/mL | <4 | <4 | <4 | <4 | <4 | <4 |
| Maximum pg/mL | 627.5 | 80.0 | 51.6 | 121.3 | 619.1 | 627.5 |

### Reference Group: All

# O. Conclusion

The results of these analytical (nonclinical) and clinical studies demonstrate that the Tosoh ST AIA-PACK BNP assay is substantially equivalent to the performance of the SIEMENS ADVIA Centaur® BNP assay.