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 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.

AI/ML Overview

The provided text describes the performance characteristics and clinical study results for the ST AIA-PACK BNP assay, an in vitro diagnostic device. This device is intended for the quantitative measurement of BNP in human K2EDTA plasma as an aid in the diagnosis of heart failure (HF).

Here's an analysis of the acceptance criteria and the study proving the device meets these criteria, based on the provided document:

1. A table of acceptance criteria and the reported device performance

The document does not explicitly state "acceptance criteria" in a table format with pass/fail thresholds. Instead, it presents performance data from various analytical and clinical studies. We can infer performance parameters that would typically be subject to acceptance criteria in such a submission.

Performance Characteristic	Acceptance Criteria (Inferred)	Reported Device Performance
Analytical Performance
Precision	CV% within acceptable range across various concentrations and sources of variation	Combined Lots (n=240):K2EDTA Plasma-1 (mean 10.588 pg/mL): Total CV 10.8%K2EDTA Plasma-2 (mean 49.873 pg/mL): Total CV 3.6%K2EDTA Plasma-3 (mean 106.718 pg/mL): Total CV 3.0%K2EDTA Plasma-4 (mean 519.429 pg/mL): Total CV 5.1%K2EDTA Plasma-5 (mean 1050.712 pg/mL): Total CV 6.1%(Detailed SD and CV% for within run, between run, between day, between lot are provided for each of 3 lots and combined data, generally showing good precision.)
Linearity/Reportable Range	Assay demonstrated to be linear over the stated range	Linear from 4.0 to 2000 pg/mL
Detection Limit (LoD)	LoD within acceptable clinical range	LoD = 1.9 pg/mL
Quantitation Limit (LoQ)	LoQ within acceptable clinical range	LoQ = 3.5 pg/mL
Analytical Specificity (Interference)	Interference due to common substances and cross-reactants < +/- 10% recovery (or other relevant thresholds)	Common Substances: Hemoglobin, Bilirubin (Unconjugated/Conjugated), Lipemia, Total Protein, Ascorbic Acid, Rheumatoid Factor, Human IgG, Cholesterol, Creatinine, Alkaline Phosphatase, HAMA IgG showed no interference (within +/- 10% recovery) at specified concentrations.Cross Reactivity: Reported % Cross Reactivity for various related peptides, generally very low.Therapeutics: 50+ therapeutic agents tested, % recovery for each was within 100+/-10% of the control.
Traceability	Demonstrated traceability to internal reference standards	Compared to internal reference standards; no international consensus reference method/material exists.
Stability	Support for stated shelf-life	Supported 12-month shelf life.
Clinical Performance
Overall Performance (at 100 pg/mL cutoff)	High sensitivity and specificity for HF diagnosis	Sensitivity: 88.4% (95% CI: 84.5-91.5%)Specificity: 70.6% (95% CI: 66.0-74.9%)PPV: 71.5%NPV: 88.0%Concordance: 78.7%AUC: 0.881

2. Sample sized used for the test set and the data provenance

Test Set Sample Size:
- Analytical Performance:
  - Precision: 6 K2EDTA plasma samples tested, each at n=80 per lot across 3 lots (total n=240 for combined lot analysis).
  - Linearity: 14 samples ranging from 3.1 - 2271.5 pg/mL.
  - Detection/Quantitation Limit: 11 low-level samples tested in 10 replicates each (2 replicates over 5 days).
  - Interference: K2EDTA samples with known BNP concentrations spiked with various interferents/cross-reactants. Specific N for each interferent test not explicitly stated but implied multiple samples.
- Clinical Performance:
  - Number of samples assayed: 825
  - Number of samples used for analysis (test set): 724 (101 excluded due to hemolysis, severe renal insufficiency, etc.)
Data Provenance:
- Country of Origin: Not explicitly stated for analytical samples. For clinical study: Samples were collected from patients presenting to Emergency Departments (EDs) at 8 clinical sites. The specific country is not mentioned, but given the FDA submission, it is likely the US or a region adhering to similar clinical trial standards.
- Retrospective or Prospective:
  - Analytical performance: Appears to be prospective testing (e.g., precision study conducted at one site with specified reagents/analyzers, linearity study explicitly designed).
  - Clinical Performance: Prospective study, as it states "The prospective study enrolled male and female patients from 8 clinical sites comprised of Emergency Departments (ED)."

3. Number of experts used to establish the ground truth for the test set and the qualifications of those experts

Number of Experts: An independent central adjudication panel comprised of 4 expert cardiologists and 1 ER physician.
Qualifications of Experts: Expert Cardiologists and an ER Physician. Their specific years of experience are not stated, but their designation as "expert" and the role in an adjudication panel for a clinical trial implies significant experience and qualifications in their respective fields related to heart failure diagnosis.

4. Adjudication method for the test set

Adjudication Method: Diagnosis of HF or non-HF was determined by an independent central adjudication panel. The panel had access to patient CRFs and clinical information (including echocardiography, other cardiac/thoracic imaging, and standard of care BNP or NT-proBNP results if available). They were blinded to the attending physician's final diagnosis and NYHA classification. The document states that the adjudication was done "to ensure standardization and accuracy of diagnosis per 2013 ACCF/AHA Guidelines for Management of HF." This indicates a consensus-based approach based on comprehensive clinical data, supervised by multiple blinded experts.

5. If a multi reader multi case (MRMC) comparative effectiveness study was done, If so, what was the effect size of how much human readers improve with AI vs without AI assistance

No, an MRMC comparative effectiveness study was not done. This submission is for an in vitro diagnostic (IVD) device (a blood test for BNP), not an AI-assisted diagnostic imaging device. Therefore, the concept of "human readers improving with AI vs without AI assistance" does not apply in this context. The study evaluates the performance of the assay itself compared to a ground truth established by clinical adjudication.

6. If a standalone (i.e. algorithm only without human-in-the-loop performance) was done

Yes, a standalone performance was done. The clinical study evaluates the performance of the "ST AIA-PACK BNP assay" itself to quantitatively measure BNP and its ability to aid in the diagnosis of HF. The results (sensitivity, specificity, AUC) are presented as the performance of the device at the given cutoff, without human interpretation of the device's output influencing the performance metrics being reported. The human element (adjudication panel) was used to establish the ground truth for diagnosis, against which the device's measurements were compared.

7. The type of ground truth used (expert consensus, pathology, outcomes data, etc.)

Type of Ground Truth: The ground truth for the clinical study was established by expert consensus based on comprehensive clinical information and per established guidelines. Specifically, the "Diagnosis of HF or non- HF was determined by an independent central adjudication panel (comprised of 4 expert cardiologists and 1 ER physician) ... per 2013 ACCF/AHA Guidelines for Management of HF." They had access to patient CRFs, echocardiography, other cardiac/thoracic imaging, and standard of care BNP/NT-proBNP results. This represents a robust form of expert consensus based on extensive clinical data.

8. The sample size for the training set

The document does not explicitly mention a separate "training set" for the device's development. The "Performance Characteristics" section details analytical and clinical validation studies. For a quantitative IVD like this, the "training" aspect is typically related to the assay's development, calibration, and optimization of reagents and protocols, rather than an explicit "training set" used in machine learning. The clinical study described is a validation study (test set).

9. How the ground truth for the training set was established

As a specific "training set" for algorithmic learning is not mentioned (as this is a biochemical assay), the concept of ground truth for a training set in that sense does not apply directly. However, the development of such an assay involves extensive work to ensure its accuracy and reliability across the dynamic range, linearity, precision, and minimizing interference. This often uses well-characterized, sometimes synthetic or spiked, samples with known concentrations to inform the assay's design and calibration. The "Traceability" section mentions that "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." This implies the "ground truth" for the internal calibration and ongoing quality control of the assay is established through gravimetric preparation and comparison to internal reference standards.

Summary

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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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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 (DICE@fda.hhs.gov) or phone (1-800-638-2041 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)

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

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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.6000 Shoreline Court, Suite 101South San Francisco, CA 94080
Contact person:	Dave WurtzSr. Director Quality, Regulatory and Clinical(415) 635-4762dave.wurtz@tosoh.com

F. Application correspondent:

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

Contact person: Stacey Dolan Sr. Manager, Regulatory Affairs (610) 240-3843 dolans@fdi.com
Summary preparation date: August 20, 2020

G. Proprietary/Established and Common Names:

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

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H. Regulatory Information:

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

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

J. Device Description:

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
1. Predicate 510(k) number(s): K031038
1. Comparison with predicate:

Similarities
	ST AIA-PACK BNP(Proposed Device)	SIEMENS ADVIA Centaur® BNP(Predicate Device)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 BNPassay is designed for IN VITRODIAGNOSTIC USE ONLY for thequantitative measurement of BNPin human (K₂EDTA) plasma onTosoh AIA System Analyzers. BNPis used as an aid in the diagnosis ofheart failure in patients presenting	For in vitro diagnostic use in thequantitative determination of B-type Natriuretic Peptide (BNP) inhuman plasma using the ADVIACentaur and ADVIA Centaur XPsystems. This assay is indicatedfor the measurement of plasmaBNP as an aid in the diagnosis

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

Differences
	ST AIA-PACK BNP(Proposed Device)	SIEMENS ADVIA Centaur® BNP(Predicate Device)K031038
Instrument System	Tosoh AIA Analyzer 2000	ADVIA Centaur and ADVIACentaur 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 labeledmouse monoclonal (mAb KY-BNP-II) anti-human BNP (Fab')2fragment specific to the ringstructure of BNP. Labeling wasdone by introducing a malemidegroup at the hinge portion of theFab'2 fragment and reacting it withalkaline phosphatase.	Acridinium ester labeled mousemonoclonal (mAb KY-BNP-II) anti-human BNP (Fab')2 fragmentspecific to the ring structure ofBNP.
Solid phase antibody	Mouse monoclonal anti-humanBNP (intact) antibody (mAb BC-203) specific to the C-terminalportion of BNP, which isimmobilized on magnetic beads	Biotinylated monoclonal mouseanti-human antibody (mAb BC-203) specific to the C-terminalportion of BNP which is coupled tostreptavidin 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° CSecond - 2.5 minutes at 37° C
Reaction	Unbound enzyme-labeledmonoclonal antibody is washedaway and a fluorogenic substrate,4-methylumbelliferyl phosphate(4MUP) is added. The degree offluorescence is directly	Following the second incubation,the unbound antibody conjugatesare washed away. An immune-complex is formed between theBNP in the sample and the twoantibody conjugates. The amountof relative light units detected by

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proportional to the amount of BNP in the sample.	the system is directly proportional to the amount of BNP in the sample.
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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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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	ApproximateConcentration (pg/mL)	Sample Composition
K2EDTA Plasma-1	10	Apparently healthypatient plasma sample
K2EDTA Plasma-2	50	Apparently healthypatient plasma sample +patient plasma sampleexpressing elevatedBNP
K2EDTA Plasma-3	100	Apparently healthypatient plasma sample +patient plasma sampleexpressing elevatedBNP
K2EDTA Plasma-4	500	Apparently healthypatient plasma sampleaugmented with BNPantigen
K2EDTA Plasma-5	1000	Apparently healthypatient plasma sampleaugmented with BNPantigen

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		K2EDTAPlasma-1	K2EDTAPlasma-2	K2EDTAPlasma-3	K2EDTAPlasma-4	K2EDTAPlasma-5
Mean Conc.(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
BetweenRun	SD	0.000	0.000	0.000	2.304	12.679
	%CV	0.0	0.0	0.0	0.4	1.2
BetweenDay	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	K2EDTAPlasma-1	K2EDTAPlasma-2	K2EDTAPlasma-3	K2EDTAPlasma-4	K2EDTAPlasma-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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Sample		K2EDTAPlasma-1	K2EDTAPlasma-2	K2EDTAPlasma-3	K2EDTAPlasma-4	K2EDTAPlasma-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
BetweenRun	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(pg/mL)	Source of Variation	Pooled Standard Deviation	%CV
K2EDTAPlasma-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
K2EDTAPlasma-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
K2EDTAPlasma-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
K2EDTAPlasma-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
K2EDTAPlasma-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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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		ExpectedValue	Mean	SD	CV(%)	Recovery(%)
No.	Low : High	(pg/mL)	(pg/mL)
	LowUndiluted
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
	HighUndiluted
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(pg/mL)	% CrossReactivity
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 LevelTarget100 pg/ml% recovery	High LevelTarget300-500 pg/ml% recovery
Acetaminophen (4-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 LevelTarget100 pg/ml% recovery	High LevelTarget300-500 pg/ml% 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	0.0039mg/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 (hydrolyzedfrom 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	0.0429mg/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 LevelTarget100 pg/ml% recovery	High LevelTarget300-500 pg/ml% 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(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(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 +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 LikelihoodRatio (PLR), CI	3.012	2.572	3.527
Negative LikelihoodRatio (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.

Regulation Number and Section

§ 862.1117 B-type natriuretic peptide test system.

(a)
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.(b)
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.”