VITROS Immunodiagnostic Products NT-proBNP II Reagent Pack

K201312 · Ortho Clinical Diagnostics · NBC · Oct 4, 2021 · Clinical Chemistry

Device Facts

• Record ID: K201312
• Device Name: VITROS Immunodiagnostic Products NT-proBNP II Reagent Pack
• Applicant: Ortho Clinical Diagnostics
• Product Code: NBC · Clinical Chemistry
• Decision Date: Oct 4, 2021
• Decision: SESE
• Submission Type: Traditional
• Regulation: 21 CFR 862.1117
• Device Class: Class 2

Intended Use

For the quantitative measurement of N-terminal pro Brain Natriuretic Peptide (NT-proBNP) in human serum and plasma (K2 EDTA or lithium heparin) using the VITROS 3600 Immunodiagnostic System to aid in the diagnosis of heart failure. The test can also be used in the assessment of heart failure severity in patients diagnosed with heart failure.

Device Story

VITROS NT-proBNP II assay is a quantitative sandwich immunoassay performed on the VITROS 3600 Immunodiagnostic System. Input: human serum or plasma (K2 EDTA or lithium heparin). Principle: one-step immunometric bridging assay; patient sample dispensed into antibody-coated well with HRP-labeled conjugate and biotinylated capture antibody; bound HRP conjugate measured via luminescent reaction (luminogenic substrate oxidation). Output: light signal proportional to NT-proBNP concentration (pg/mL). Used in clinical laboratories; operated by trained personnel. Results interpreted against age-dependent cutoffs (ED setting) or a single cutoff (outpatient setting) to aid heart failure diagnosis and severity assessment. Benefits: provides objective biomarker data to assist clinicians in diagnosing heart failure and assessing disease severity, potentially improving diagnostic accuracy and patient management.

Clinical Evidence

Clinical performance was established in a multi-center prospective study of 2,200 ED subjects and 777 outpatient subjects. Primary endpoints included AUC for heart failure diagnosis and correlation with NYHA functional classification. AUC for ED subjects ranged from 0.904 to 0.954 across subgroups. The test demonstrated a statistically significant relationship between NT-proBNP levels and NYHA class (p < 0.0001).

Technological Characteristics

Sandwich immunoassay; mouse monoclonal anti-NT-proBNP antibody coated wells; HRP-conjugated sheep monoclonal anti-NT-proBNP antibody; luminescent detection; measuring range 20-30,000 pg/mL. Requires VITROS 3600 Immunodiagnostic System. Software-controlled automated processing. No specific material standards (e.g., ASTM) cited for construction.

Indications for Use

Indicated for quantitative measurement of NT-proBNP in human serum/plasma to aid in diagnosis and severity assessment of heart failure in patients aged 22 years and older.

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

• Roche Elecsys® proBNP II Immunoassay (K072437)

Submission Summary (Full Text)

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Image /page/0/Picture/0 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.

October 4, 2021

Ortho Clinical Diagnostics Ann Quinn Director, Regulatory Affairs 100 Indigo Creek Drive Rochester, New York 14626

Re: K201312

Trade/Device Name: VITROS® Immunodiagnostic Products NT-proBNP II Reagent Pack Regulation Number: 21 CFR 862.1117 Regulation Name: B-Type Natriuretic Peptide Test System Regulatory Class: Class II Product Code: NBC Dated: November 6, 2020 Received: November 9, 2020

Dear Ann Quinn:

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.

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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 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 (QS) regulation (21 CFR Part 820) for devices or current good manufacturing practices (21 CFR 4, Subpart A) for combination products; and, if applicable, the electronic product radiation control provisions (Sections 531-542 of the Act); 21 CFR 1000-1050.

Also, please note the regulation entitled, "Misbranding by reference to premarket notification" (21 CFR Part 807.97). For questions regarding the reporting of adverse events under the MDR regulation (21 CFR Part 803), please go to 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. 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

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# Indications for Use

510(k) Number (if known) K201312

Device Name

VITROS® Immunodiagnostic Products NT-proBNP II Reagent Pack

Indications for Use (Describe) For in vitro diagnostic use only.

For the quantitative measurement of N-terminal pro Brain Natriuretic Peptide (NT-proBNP) in human serum and plasma (K2 EDTA or Lithium Heparin) using the VITROS 3600 Immunodiagnostic System to aid in the diagnosis of heart failure. The test can also be used in the assessment of heart failure severity in patients diagnosed with heart failure.

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

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This summary of 510(k) safety and effectiveness information is being submitted in accordance with the requirements of SMDA 1990 and 21 CFR 807.92.

The assigned 510(k) number is: K201312

- 1. Submitter Ortho-Clinical Diagnostics, Inc. name, 100 Indigo Creek Drive Rochester, NY 14626 address, contact P: [phone] F: [phone] Contact Person: Ann Quinn, Director, Regulatory Affairs
- September 23, 2021 2. Preparation Date
- 3. Device Trade or Proprietary Names: VITROS® Immunodiagnostic Products NT-proBNP II Reagent Pack name Common Name: VITROS NT-proBNP II Classification: B-Type natriuretic peptide test system (862.1117) Class II Product Code: NBC
- Roche Elecsys® proBNP II Immunoassay, K072437 4. Predicate Device
- Device 5. The VITROS NT-proBNP II test is performed using the VITROS description VITROS NT-proBNP II Reagent Pack and the VITROS NT-proBNP II Calibrators on the VITROS Systems.

The VITROS NT-proBNP II test utilizes a one-step immunometric bridging assay design. A well is pushed from the pack and patient sample is dispensed into the antibody coated well. The assay reagent and the conjugate reagent are then dispensed into the well with the patient sample. NT-proBNP present in the sample binds with horseradish peroxidase (HRP)-labeled antibody conjugate which is captured by biotinylated anti-NT-proBNP capture antibody which is bound to Streptavidin coated

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| | microwells. The well is incubated for 8 minutes, before unbound<br>materials are removed by washing. |
|------------------------------|-------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------|
| | The bound HRP conjugate is measured by a luminescent reaction. A<br>reagent containing luminogenic substrate (a luminol derivative and a<br>peracid salt) and an electron transfer agent, is added to the wells. The HRP<br>in the bound conjugate catalyzes the oxidation of the luminol derivative,<br>producing light. The electron transfer agent (a substituted acetanilide)<br>increases the level of light produced and prolongs its emission. The light<br>signals are read by the System. The amount of HRP conjugate bound is<br>directly proportional to the concentration of NT-proBNP present. |
| 6. Device<br>intended<br>use | Rx ONLY<br>For <i>in vitro</i> diagnostic use only. |
| | For the quantitative measurement of N-terminal pro Brain Natriuretic<br>Peptide (NT-proBNP) in human serum and plasma (K2 EDTA or Lithium<br>Heparin) using the VITROS 3600 Immunodiagnostic System to aid in the<br>diagnosis of heart failure. The test can also be used in the assessment of<br>heart failure severity in patients diagnosed with heart failure. |

- 7. Compari-The following tables provide a summary of the key features of the new son to device assessed against the predicate. predicate device:

| Device<br>Characteristic | New Device<br>VITROS NT-proBNP II | Predicate Device<br>Roche Elecsys proBNP<br>II Immunoassay,<br>K072437 | |
|--------------------------|--------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------|---------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------|-----------------------------------------------------------------------------------|
| Intended Use | Rx ONLY<br>For <i>in vitro</i> diagnostic use only.<br><br>For the quantitative measurement of N-terminal pro Brain<br>Natriuretic Peptide (NT-proBNP) in human serum and<br>plasma (K2 EDTA or Lithium Heparin) using the VITROS<br>3600 Immunodiagnostic System to aid in the diagnosis of<br>heart failure. The test can also be used in the assessment of<br>heart failure severity in patients diagnosed with heart<br>failure. | For the quantitative<br>determination of N-<br>terminal pro-Brain<br>natriuretic peptide in<br>human serum and<br>plasma. Elecsys proBNP<br>II assay is used as an aid<br>in the diagnosis of<br>individuals suspected of<br>having congestive heart<br>failure. The test is<br>further indicated for the<br>risk stratification of<br>patients with acute<br>coronary syndrome and<br>congestive heart failure.<br>The test may also serve | |
| Device<br>Characteristic | New Device<br>VITROS NT-proBNP II | Predicate Device<br>Roche Elecsys proBNP<br>II Immunoassay,<br>K072437 | |
| Intended Use | See above | as an aid in the<br>assessment of increased<br>risk of cardiovascular<br>events and mortality in<br>patients at risk for heart<br>failure who have stable<br>coronary artery disease.<br>The electro-<br>chemiluminescense<br>immunoassay "ECLIA"<br>is intended for use on<br>Elecsys and cobas e<br>immunoassay analyzers. | |
| Basic Principle | Sandwich immunoassay | Same | |
| Antibody | Monoclonal anti-NT-proBNP | Same | |
| Sample Type | Serum and plasma | Same | |
| Measuring<br>Range | 20-30,000 pg/mL | 5-35,000 pg/mL | |
| Traceability | Standardized against the Elecsys proBNP II assay. | Standardized against the<br>Elecsys proBNP assay.<br><75 years: 125 pg/mL<br>≥75 years: 450 pg/mL | |
| Result<br>Interpretation | For patients presenting to the ED settings with acute or<br>worsening dyspnea and clinical suspicion of HF, the<br>VITROS NT-proBNP II test results should be interpreted as<br>indicated in the table below. | | |
| | VITROS NT-proBNP II Test Results<br>(pg/mL) | Age Group (Years) | Interpretation of Results |
| | <300 | All | Negative: Heart Failure Unlikely |
| | ≥300 to <450 | 22-<50 | Gray Zone: Result Indeterminate -<br>Consider other causes of NT-proBNP elevation |
| | ≥300 to <900 | 50-<75 | |
| | ≥300 to <1800 | ≥75 | |
| | ≥450 | 22-<50 | Positive: Heart Failure Likely |
| | ≥900 | 50-<75 | |
| | ≥1800 | ≥75 | |

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| Device<br>Characteristic | New Device<br>VITROS NT-proBNP II | Predicate Device<br>Roche Elecsys proBNP<br>II Immunoassay,<br>K072437 | | | | | | | | | |
|--------------------------|----------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------|------------------------------------------------------------------------|--|--|--|--|--|--|--|--|--|
| | For ambulatory patients presenting to outpatient facilities<br>with clinical suspicion of HF not previously diagnosed and<br>at least one sign, symptom or risk factor for HF, the<br>VITROS NT-proBNP II test results should be interpreted as<br>indicated in the table below. | | | | | | | | | | |
| | VITROS NT-proBNP II Test Results<br>(pg/mL) Age Group Interpretation of Results <125 All Negative: Heart Failure Unlikely ≥125 All Consider Heart Failure as well as other<br>causes* of NT-proBNP elevation. | | | | | | | | | | |

#### 8. Nonclinical performance

Several nonclinical tests were performed.

### Precision

Precision was evaluated consistent with CLSI document EP05-A3, Evaluation of Precision of Quantitative Measurement Procedures; Approved Guideline—Third Edition. Two replicates of each of eleven fluids, eight human individual or serum pools and three controls, were tested on two separate occasions per day on at least 20 different test days. The experiment was performed using three Master Lots on one VITROS 3600 Immunodiagnostic System. The data presented are a representation of the product performance.

| Conc. Units - pg/mL | | | | | | | | |
|---------------------|-------------|-----|--------------|-----|---------------|-----|---------------------|-------------|
| NT-proBNP | Within-run* | | Within-cal** | | Within-lab*** | | No.<br>Observations | No.<br>Days |
| Conc. | SD | %CV | SD | %CV | SD | %CV | | |
| 33.6 | 0.52 | 1.5 | 1.63 | 4.9 | 1.91 | 5.7 | 80 | 20 |
| 70.3 | 1.45 | 2.1 | 3.31 | 4.7 | 3.87 | 5.5 | 80 | 20 |
| 97.8 | 1.45 | 1.5 | 3.90 | 4.0 | 4.62 | 4.7 | 80 | 20 |
| 214 | 3.9 | 1.8 | 6.4 | 3.0 | 7.9 | 3.7 | 80 | 20 |
| 365 | 4.4 | 1.2 | 10.9 | 3.0 | 14.0 | 3.8 | 80 | 20 |
| 815 | 11.6 | 1.4 | 28.7 | 3.5 | 30.5 | 3.7 | 80 | 20 |
| 937 | 10.1 | 1.1 | 26.6 | 2.8 | 29.6 | 3.2 | 80 | 20 |
| 1730 | 19 | 1.1 | 86 | 5.0 | 84 | 4.8 | 80 | 20 |

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| Conc. Units - pg/mL | | | | | | | No. | No. |
|---------------------|-------------|-----|--------------|-----|---------------|-----|--------------|------|
| NT-proBNP<br>Conc. | Within-run* | | Within-cal** | | Within-lab*** | | Observations | Days |
| | SD | %CV | SD | %CV | SD | %CV | | |
| 5830 | 80 | 1.4 | 155 | 2.6 | 159 | 2.7 | 80 | 20 |
| 11300 | 110 | 1.0 | 210 | 1.9 | 270 | 2.4 | 80 | 20 |
| 24600 | 370 | 1.5 | 710 | 2.9 | 730 | 3.0 | 80 | 20 |

* Within-run (repeatability). Between Duplicate precision averaged over all runs.

** Within-calibration. Total precision with weighted components of within-run, between-run, and between-day variation.

*** Within-lab. A measure of the effect of recalibration on total precision, calculated within reagent lot, using data from at least 4 calibrations.

An additional analysis was conducted to evaluate total imprecision. The data presented are a representation of the product performance.

| System | Mean<br>pg/mL | Between Lot* | | Between Day** | | Between<br>Runs*** | | Within Run /<br>Residual**** | | Total***** | | Observation |
|--------|---------------|--------------|--------|---------------|--------|--------------------|--------|------------------------------|--------|------------|--------|-------------|
| | | SD | CV (%) | SD | CV (%) | SD | CV (%) | SD | CV (%) | SD | CV (%) | |
| 3600 | 32 | 2.34 | 7.32 | 0.875 | 2.74 | 1.28 | 4.02 | 0.545 | 1.71 | 2.86 | 8.96 | 240 |
| | 69.5 | 3.92 | 5.65 | 1.89 | 2.71 | 1.83 | 2.63 | 1.58 | 2.27 | 4.98 | 7.17 | 240 |
| | 97 | 4.65 | 4.79 | 2.65 | 2.73 | 1.89 | 1.95 | 1.56 | 1.61 | 5.89 | 6.07 | 240 |
| | 369 | 9.14 | 2.48 | 6.32 | 1.71 | 7.75 | 2.10 | 4.90 | 1.33 | 14.4 | 3.91 | 240 |
| | 823 | 15.9 | 1.94 | 11.6 | 1.40 | 20.5 | 2.49 | 12.5 | 1.52 | 31.0 | 3.77 | 240 |
| | 949 | 26.6 | 2.81 | 17.5 | 1.84 | 13.9 | 1.46 | 12.2 | 1.29 | 36.8 | 3.88 | 240 |
| | 1745 | 10.8 | 0.62 | 44.5 | 2.55 | 56.9 | 3.26 | 20.3 | 1.16 | 75.8 | 4.34 | 240 |
| | 5859 | 75 | 1.28 | 92.1 | 1.57 | 55.4 | 0.95 | 80.3 | 1.37 | 154 | 2.62 | 240 |
| | 24686 | 329 | 1.33 | 355 | 1.44 | 455 | 1.84 | 371 | 1.50 | 761 | 3.08 | 240 |
| | 214 | 6.78 | 3.17 | 3.66 | 1.71 | 3.11 | 1.45 | 3.50 | 1.64 | 9.02 | 4.22 | 240 |
| | 11392 | 206 | 1.81 | 145 | 1.28 | 65.2 | 0.57 | 176 | 1.54 | 314 | 2.76 | 240 |

*Between lot: Variability of the test performance from lot to lot.

**Between day: Variability of the test performance from day to day.

*** Between run: Variability of the test performance from run to run.

*** Within Run / Residual Variability.

****Total: Variability of the test incorporating factors of Instrument, lot, day and run.

# Limit of Detection

Detection studies for the VITROS NT-proBNP II test were evaluated according to CLSI document EP17-A2, Evaluation of Detection Capability for Clinical Laboratory Measurement Procedures; Approved Guideline - Second Edition.

The Limit of Detection (LoD) for the VITROS NT-proBNP II test was designed to be less than or equal to 30.0 pg/mL. The observed LoD was determined to be 0.46 pg/mL. The claimed LoD is 0.49 pg/mL. The Limit of Quantitation (LoQ) for the VITROS NT-

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proBNP II test was designed to be less than or equal to 30.0 pg/mL at 20% CV. The observed LoQ at 20% CV was determined to be 0.46 pg/mL. The claimed LoQ is set at 20.0 pg/mL to maintain linearity within the measuring range.

| LoD | LoQ |
|-------|-------|
| pg/mL | pg/mL |
| 0.49 | 20.0 |

# Limit of Detection and Limit of Quantitation

# Linearity

Linearity studies were performed according to CLSI document EP06-A, Evaluation of the Linearity of Quantitative Measurement Procedures: A Statistical Approach: Approved Guideline. Three Master Lots of VITROS NT-proBNP II test were tested on the VITROS 3600 Immunodiagnostic System. A low and high sample pool was prepared and mixed to give fourteen (14) further pools of intermediate concentrations. The low and high linearity pools and the interim dilutions between the low and high linearity pools were assayed in triplicate. The VITROS NT-proBNP II test was linear over the measuring range, from 20.0 to 30,000 pg/mL for the VITROS 3600 Immunodiagnostic System.

# Matrix Comparison

A total of 160 unaltered matched serum, EDTA plasma and lithium heparin plasma samples covering the measuring range of the VITROS NT-proBNP II test were used to assess sample matrix differences between serum, EDTA plasma, and lithium heparin plasma samples.

Samples were collected into each of the following tube types: serum plus clot activator plastic tube (serum), K2-EDTA plastic tube (EDTA), and lithium heparin plasma plastic tube (Li-Hep). Each serum sample was tested in duplicate while the Li-Hep plasma and EDTA plasma samples were assayed in singleton using the VITROS 3600 Immunodiagnostic System.

Overall, the results show EDTA and Li-Hep plasma plastic collection tubes did not show a significant effect on the test results when compared to serum and met the acceptance criteria. All three tube types, serum, EDTA plasma, and Li-Hep plasma are suitable for use with the VITROS NT-proBNP II test.

# Analytical Specificity

The VITROS NT-proBNP II test was evaluated for interference according to CLSI guidelines EP07 3rd ed - Interference Testing in Clinical Chemistry. Commonly encountered substances were tested on three Master Lots of reagents. Of the compounds tested in the table below, none was found to cause a bias of >10% with the test at the concentrations indicated at nominal NT-proBNP concentrations of 125 pg/mL (14.8 pmol/L) and 2000 pg/mL (236 pmol/L).

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| Compound | Concentration | | Compound | Concentration | |
|------------------------------------------------------------|---------------|-------------|-----------------------------|---------------|-------------|
| Acetaminophen | 156 µg/mL | 1030 µmol/L | Ibuprofen | 21.9 mg/dL | 1.06 mmol/L |
| Acetylcysteine | 15.0 mg/dL | 920 µmol/L | Insulin | 3.12 µg/dL | 5.37 nmol/L |
| Adrenaline (Epinephrine) | 20.0 µg/dL | 1.09 µmol/L | Intralipid | 2.00 g/dL | NA |
| Alprazolam | 25.8 µg/dL | 836 nmol/L | L-dopa (Levodopa) | 750 µg/dL | 38.0 µmol/L |
| Amlodipine besylate | 10.5 µg/dL | 184 nmol/L | Levothyroxine | 42.9 µg/dL | 552 nmol/L |
| Amoxicillin | 5.40 mg/dL | 148 µmol/L | Lidocaine | 1.50 mg/dL | 64.0 µmol/L |
| Ascorbic acid | 5.25 mg/dL | 298 µmol/L | Methyldopa sesquihydrate | 2.25 mg/dL | 94.4 µmol/L |
| Atorvastatin<br>calciumtrihydrate | 162 µg/dL | 1.34 µmol/L | Methylprednisolone | 783 µg/dL | 20.9 µmol/L |
| Benazepril HCl | 44.0 µg/dL | 955 nmol/L | Metoprolol hemitartrate | 150 µg/dL | 2.19 µmol/L |
| Bilirubin, conjugated | 40.0 mg/dL | 474 µmol/L | Metronidazole | 12.3 mg/dL | 718 µmol/L |
| Bilirubin, unconjugated | 40.0 mg/dL | 684 µmol/L | Molsidomine | 18.0 µg/dL | 743 nmol/L |
| Biotin | 3510 ng/mL | 14.4 µmol/L | Naproxen sodium | 39.3 mg/dL | 1.56 mmol/L |
| Caffeine | 10.8 mg/dL | 556 µmol/L | Nicardipine HCL | 46.5 µg/dL | 901 nmol/L |
| Carvedilol | 43.2 µg/dL | 1.06 µmol/L | Nifedipine | 58.8 µg/dL | 1.70 µmol/L |
| Ceftriaxone<br>disodium<br>hemi(heptahydrate) | 100 mg/dL | 1510 µmol/L | Omeprazole | 840 µg/dL | 24.3 µmol/L |
| Cholesterol | 400 mg/dL | 10.3 mmol/L | Oxycodone HCl | 32.4 µg/dL | 0.92 µmol/L |
| Clopidogrel<br>hydrogensulfate | 2.40 µg/dL | 57.2 nmol/L | Phenobarbital | 69.0 mg/dL | 2.97 mmol/L |
| Cotinine | 240 µg/dL | 13.6 µmol/L | Phenprocoumo<br>n(Marcumar) | 1.50 mg/dL | 53.5 µmol/L |
| Creatinine | 15.0 mg/dL | 1.33 mmol/L | Propafenone HCL | 72.0 µg/dL | 1.91 µmol/L |
| Cyclosporine | 180 µg/dL | 1.50 µmol/L | Pseudoephedrine HCl | 330 µg/dL | 16.4 µmol/L |
| Dextran | 2.40 g/dL | 600 µmol/L | Rheumatoid Factor | 1500 IU/mL | NA |
| Digitoxin | 7.50 µg/dL | 98.0 nmol/L | Rifampicin (Rifampin) | 4.80 mg/dL | 58.3 µmol/L |
| Digoxin | 3.90 µg/dL | 49.9 nmol/L | Salicylic acid | 2.86 mg/dL | 207 µmol/L |
| Diphenhydramine HCl | 77.4 µg/dL | 2.65 µmol/L | Salmeterol | 1.65 µg/dL | 39.7 nmol/L |
| Dipyrone (as 4-<br>methylaminoantipyrine<br>Hydrochloride) | 3.30 mg/dL | 130 µmol/L | Sotalol hydrochloride | 510 µg/dL | 16.5 µmol/L |
| Dypyridamole | 1.00 mg/dL | 19.8 µmol/L | Spironolactone | 55.5 µg/dL | 1.33 µmol/L |
| Doxycycline hyclate | 1.80 mg/dL | 35.1 µmol/L | Streptokinase | 150,000 U/dL | NA |
| Enalaprilat dihydrate | 81.9 µg/dL | 2.13 µmol/L | Theophylline | 6.00 mg/dL | 333 µmol/L |
| Ethanol | 600 mg/dL | 130 mmol/L | Tolbutamide | 54.9 mg/dL | 2.03 mmol/L |
| Fibrinogen | 1000 mg/dL | NA | Total Protein | 15.0 g/dL | NA |
| Furosemide | 1.59 mg/dL | 48.1 µmol/L | tPA (Alteplase) | 1.20 mg/dL | NA |
| Gentamicin Sulfate | 3.51 mg/dL | 61.0 µmol/L | Triglyceride | 1500 mg/dL | 16.9 mmol/L |
| Glycerylnitrate<br>(Nitroglycerin) | 1.20 µg/dL | 52.8 nmol/L | Valproic Acid | 31.8 mg/dL | 2.21 mmol/L |
| HAMA (Human Anti-<br>MouseAntibody | 800 µg/L | NA | Vancomycin Hydrochloride | 12.3 mg/dL | 82.8 µmol/L |
| Hemoglobin | 1000 mg/dL | 155 µmol/L | Verapamil Hydrochloride | 160 µg/dL | 3.26 µmol/L |
| Heparin (Sodium), UFH | 330 U/dL | NA | Warfarin | 8.00 mg/dL | 260 µmol/L |

The substances shown to interfere with the VITROS NT-proBNP II test are provided in the table below. These results are representative. The degree of interference at concentrations other than those listed might not be predictable from these results. Other interfering substances may be encountered in the patient population.

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| Interferent | Interferent Concentration | Measured NT-proBNP Concentration* | | % Bias** |
|------------------|---------------------------|-----------------------------------|--------|----------|
| | | pg/mL | pmol/L | |
| Cefoxitin sodium | 697 mg/dL | 84.3 | 9.95 | -24.1 |
| | 15.5 mmol/L | 922 | 109 | -19.1 |
| | 311 mg/dL | 105 | 12.4 | -10.0 |
| | 306 mg/dL | 806 | 95.1 | -10.0 |
| Sodium Azide | 100 mg/dL | 91.4 | 10.8 | -12.1 |
| | 85.9 mg/dL | 93.6 | 11.0 | -10.0 |
| | 15.4 mmol/L | | | |
| | 13.2 mmol/L | | | |

* Average test replicate determinations.

** Estimate of the maximum difference observed as a percentage.

The cross-reactivity of the VITROS NT-proBNP II test was evaluated by adding the following substances to a human serum sample containing no NT-proBNP.

| Cross-Reactant | Concentration | | Mean Result of Control Sample | | Mean Result of Cross-Reactant Sample | | % Cross- Reactivity |
|---------------------------------------|----------------|--------------|-------------------------------|--------|--------------------------------------|--------|---------------------|
| | | | pg/mL | pmol/L | pg/mL | pmol/L | |
| ANP28 | 3.10 µg/mL | 1.01 nmol/L | * | * | * | * | * |
| proBNP (glycosylated) | 3000 pg/mL | N/A | -0.14 | -0.02 | 57.6 | 6.80 | 1.9 |
| proBNP (nonglycosylated) | 3000 pg/mL | 0.249 nmol/L | -0.48 | -0.06 | 863 | 102 | 28.8 |
| NT-proANP1-30<br>(preproANP25-55) | 3.50 µg/mL | 0.998 µmol/L | * | * | * | * | * |
| NT-proANP31-67<br>(preproANP56-92) | 1.00 ng/mL | 0.258 nmol/L | * | * | * | * | * |
| NT-proANP79-98<br>(preproANP 104-123) | 1.00 ng/mL | 0.458 nmol/L | * | * | * | * | * |
| BNP32 (Natrecor®) | 3.50 µg/mL | 1.01 µmol/L | * | * | * | * | * |
| CNP22 | 2.20 µg/mL | 1.00 µmol/L | * | * | * | * | * |
| Adrenomedullin | 1.00 ng/mL | 0.166 nmol/L | * | * | * | * | * |
| Aldosterone | 0.600 ng/mL | 1.66 nmol/L | * | * | * | * | * |
| Angiotensin I | 0.600 ng/mL | 0.463 nmol/L | * | * | * | * | * |
| Angiotensin II | 0.600 ng/mL | 0.574 nmol/L | * | * | * | * | * |
| Angiotensin III | 1.00 ng/mL | 1.07 nmol/L | * | * | * | * | * |
| Endothelin | 20.0 pg/mL | 8.03 pmol/L | * | * | * | * | * |
| Urodilatin | 3.50 µg/mL | 0.998 µmol/L | * | * | * | * | * |
| Arg-Vasopressin | 1.00 µg/mL | 0.922 µmol/L | * | * | * | * | * |
| Renin | 50.0 ng/mL | 28.4 nmol/L | * | * | * | * | * |

*Not Detectable (ND). Concentration was below the measuring range of the test, 20.0-30,000 pg/mL (2.36-3,540 pmol/L).

The cross-reactivity of the VITROS NT-proBNP II test was evaluated by adding the following substances to a human serum sample containing NT-proBNP at a concentration of 125 pg/mL (14.8 pmol/L).

| Cross-Reactant | Concentration | Mean NT-proBNP<br>Result of Control<br>Sample | | Mean NT-proBNP<br>Result of Cross-<br>Reactant Sample | | % Cross-<br>Reactivity |
|--------------------------|---------------|-----------------------------------------------|--------|-------------------------------------------------------|--------|------------------------|
| | | pg/mL | pmol/L | pg/mL | pmol/L | |
| ANP28 | 3.10 µg/mL | 112 | 13.2 | 113 | 13.3 | <1.0 |
| proBNP (glycosylated) | 3000 pg/mL | 105 | 12.4 | 183 | 21.6 | 2.6 |
| proBNP (nonglycosylated) | 3000 pg/mL | 118 | 13.9 | 1290 | 152 | 39.1 |

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

| NT-proANP1-30<br>(preproANP25-55) | 3.50 µg/mL | 0.998 µmol/L | 113 | 13.3 | 775 | 91.5 | <1.0 |
|--------------------------------------|-------------|--------------|-----|------|-----|------|------|
| NT-proANP31-67<br>(preproANP56-92) | 1.00 ng/mL | 0.258 nmol/L | 112 | 13.2 | 113 | 13.3 | <1.0 |
| NT-proANP79-98<br>(preproANP104-123) | 1.00 ng/mL | 0.458 nmol/L | 112 | 13.2 | 114 | 13.5 | <1.0 |
| BNP32 (Natrecor®) | 3.50 µg/mL | 1.01 µmol/L | 112 | 13.2 | 115 | 13.6 | <1.0 |
| CNP22 | 2.20 µg/mL | 1.00 µmol/L | 112 | 13.2 | 114 | 13.5 | <1.0 |
| Adrenomedullin | 1.00 ng/mL | 0.166 nmol/L | 112 | 13.2 | 114 | 13.5 | <1.0 |
| Aldosterone | 0.600 ng/mL | 1.66 nmol/L | 112 | 13.2 | 117 | 13.8 | <1.0 |
| Angiotensin I | 0.600 ng/mL | 0.463 nmol/L | 112 | 13.2 | 112 | 13.2 | <1.0 |
| Angiotensin II | 0.600 ng/mL | 0.574 nmol/L | 112 | 13.2 | 111 | 13.1 | <1.0 |
| Angiotensin III | 1.00 ng/mL | 1.07 nmol/L | 112 | 13.2 | 112 | 13.2 | <1.0 |
| Endothelin | 20.0 pg/mL | 8.03 pmol/L | 113 | 13.3 | 113 | 13.3 | <1.0 |
| Urodilatin | 3.50 µg/mL | 0.998 µmol/L | 112 | 13.2 | 114 | 13.5 | <1.0 |
| Arg-Vasopressin | 1.00 µg/mL | 0.922 µmol/L | 112 | 13.2 | 117 | 13.8 | <1.0 |
| Renin | 50.0 ng/mL | 28.4 nmol/L | 112 | 13.2 | 112 | 13.2 | <1.0 |

Cross-reactivity was expressed as the mean result obtained for the cross-reactant sample minus the mean result obtained for the control sample divided by the cross-reactant concentration in percentage terms.

# Dilution

Serum or plasma (K2 EDTA or Lithium Heparin) samples with concentrations greater than the measuring range may be automatically diluted on the system up to 10-fold (1 part sample with 9 parts diluent) by the VITROS 3600 Immunodiagnostic System with the VITROS High Sample Diluent B Reagent Pack prior to testing.

# High Dose Hook

The VITROS NT-proBNP II test has no high dose hook effect up to a concentration of 300,000 pg/mL.

# Expected Values

It is recommended that each laboratory establish its own expected values for the population it serves. The VITROS NT-proBNP II test Reference Interval (RI) was established for six subgroups, based on age and gender from the serum of 385 female and 374 male healthy donors.

Subjects were excluded if they met any of the following exclusion criteria:

- . Current smokers, subjects with cardiac conditions and disease, high blood pressure, kidney disease, diabetes, cancer within the last five years, stroke, and asthma or other lung disease within the last five years.
- Subjects who have reported high cholesterol, high triglycerides, thyroid disease, and females who are pregnant.
- Additional exclusion criteria:
- Troponin > 99th percentile (> 0.034 ng/mL VITROS Troponin I ES assay) .
- HbA1c > 6.5% .
- Creatinine (eGFR ≤ 60 mL/min) ●

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

| Age | Gender | n | RI Lower<br>Limit<br>(pg/mL) | RI Upper<br>Limit<br>(pg/mL) |
|-----------|--------|-----|------------------------------|------------------------------|
| 22 - <50 | Female | 129 | <20.0 | 95.3 |
| 50 - < 75 | Female | 127 | <20.0 | 221 |
| ≥75 | Female | 129 | <20.0 | 296 |
| 22 - <50 | Male | 131 | <20.0 | 125 |
| 50 - < 75 | Male | 120 | <20.0 | 299 |
| ≥75 | Male | 123 | <20.0 | 326 |
| Overall | | 756 | <20.0 | 217 |

The reference interval was conducted in accordance with the CLSI EP28. Analysis at the 95% confidence level vields the ranges shown in the table.

#### 9. Clinical performance

The clinical performance information should only be used as a guide. It is recommended that each laboratory determine and confirm the diagnostic cutoffs for the population it serves.

# Aid in Diagnosis of Heart Failure

# Emergency Department Setting

A multi-center prospective study including 20 collection sites across the United States was conducted to establish the performance characteristics of the VITROS NT-proBNP II test. Subjects 22 years and older presenting to the Emergency Department (ED) with dyspnea (acute or worsening) and clinical suspicion of heart failure (HF) were enrolled into the study. Subjects with terminal kidney failure on chronic dialysis and subjects with dyspnea clearly not secondary to HF were excluded from the study. The final clinical diagnosis was adjudicated by independent cardiologists or ED physicians experienced in diagnosing HF. Individuals in the population were African American (36.55%) and Caucasian (59.59%), withthe remaining 3.86% represented by other races. Dyspnea was acute in 44.09%, worsening in 55.45% and not specified in 0.45% of subjects.

The VITROS NT-proBNP II test results were determined from 2200 ED subjects, 1016 (46.18%) females and 1184 (53.82%) males, ranging in age from 22 to 106 years. The descriptive statistics for the VITROS NT-proBNP II test results(pg/mL) were determined within and across gender by age group and are summarized in the following tables:

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

#### All Subjects

| Study<br>Population | Heart Failure | | | | Non-Heart Failure | | | |
|---------------------|---------------|--------|-------|--------|-------------------|--------|-------|-------|
| Age (years) | 22-<50 | 50-<75 | ≥75 | All | 22-<50 | 50-<75 | ≥75 | All |
| N | 114 | 538 | 443 | 1095 | 141 | 630 | 334 | 1105 |
| Mean | 6840 | 6510 | 7810 | 7070 | 261 | 694 | 1120 | 767 |
| SD | 20500 | 9490 | 10100 | 11400 | 568 | 1720 | 1690 | 1630 |
| Median | 2150 | 3550 | 4850 | 3780 | 58.4 | 177 | 473 | 247 |
| Min | 20.0 | 53.7 | 295 | 20.0 | 20.0 | 20.0 | 20.0 | 20.0 |
| Max | 178000 | 86300 | 94600 | 178000 | 4710 | 23800 | 13600 | 23800 |

#### Female Subjects

| Study<br>Population | Heart Failure | | | | Non-Heart Failure | | | |
|---------------------|---------------|--------|-------|--------|-------------------|--------|------|-------|
| Age (years) | 22-<50 | 50-<75 | ≥75 | All | 22-<50 | 50-<75 | ≥75 | All |
| N | 43 | 194 | 217 | 454 | 71 | 306 | 185 | 562 |
| Mean | 10300 | 6830 | 7790 | 7620 | 196 | 648 | 948 | 690 |
| SD | 32200 | 10000 | 10600 | 13900 | 356 | 1800 | 1280 | 1540 |
| Median | 1920 | 3620 | 4710 | 3850 | 68.2 | 157 | 444 | 226 |
| Min | 20.0 | 102 | 296 | 20.0 | 20.0 | 20.0 | 20.0 | 20.0 |
| Max | 178000 | 86300 | 94600 | 178000 | 2010 | 23800 | 7870 | 23800 |

#### Male Subjects

| Study<br>Population | Heart Failure | | | | Non-Heart Failure | | | |
|---------------------|---------------|--------|-------|-------|-------------------|--------|-------|-------|
| Age (years) | 22-<50 | 50-<75 | ≥75 | All | 22-<50 | 50-<75 | ≥75 | All |
| N | 71 | 344 | 226 | 641 | 70 | 324 | 149 | 543 |
| Mean | 4720 | 6330 | 7830 | 6680 | 326 | 737 | 1330 | 846 |
| SD | 6780 | 9170 | 9730 | 9180 | 718 | 1640 | 2080 | 1720 |
| Median | 2260 | 3480 | 5020 | 3690 | 43.6 | 203 | 519 | 275 |
| Min | 195 | 53.7 | 295 | 53.7 | 20.0 | 20.0 | 20.0 | 20.0 |
| Max | 39200 | 70400 | 86000 | 86000 | 4710 | 17100 | 13600 | 17100 |

The area under the Receiver Operating Characteristic (ROC) curve (AUC) with a 95% confidence interval (CI) for the VITROS NT-proBNP II test within and across age groups within and across gender are presented in the table below. The AUC ranged between 0.904 to 0.954 within and across gender.

| Age Group | All Subjects | | Female Subjects | | Male Subjects | |
|--------------|--------------|----------------------------|-----------------|----------------------------|---------------|----------------------------|
| | AUC | 95% Confidence<br>Interval | AUC | 95% Confidence<br>Interval | AUC | 95% Confidence<br>Interval |
| 22-<50 years | 0.954 | 0.928-0.979 | 0.952 | 0.905-0.999 | 0.947 | 0.912-0.983 |
| 50-<75 years | 0.922 | 0.907-0.937 | 0.926 | 0.904-0.948 | 0.917 | 0.896-0.938 |
| ≥75 years | 0.915 | 0.895-0.934 | 0.920 | 0.895-0.946 | 0.904 | 0.872-0.937 |
| Overall | 0.920 | 0.909-0.931 | 0.925 | 0.910-0.941 | 0.914 | 0.898-0.930 |

AUC analyses were also performed for relevant clinical subgroups. The AUC with a 95% confidence interval (CI) for the VITROS NT-proBNP II test across gender within the relevant clinical subgroups are presented in the table below. The AUCof the VITROS NT-proBNP II test for each of the relevant clinical subgroups for subjects with or without the condition was greater than or equal to 0.899.

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

| Relevant Clinical Subgroups | | | | | | |
|-----------------------------------------|-----------------------------|-------------------------|----------------------------------------|--------------------------------|-------------------------|--|
| | Subjects With the Condition | | | Subjects Without the Condition | | |
| Subgroups | AUC | 95% Confidence Interval | Subgroups | AUC | 95% Confidence Interval | |
| History of HF<br>(N=1220) | 0.899 | 0.881-0.918 | No History of HF<br>(N=970) | 0.932 | 0.917-0.947 | |
| eGFR <60*<br>mL/min/1.73 m2<br>(N=1012) | 0.900 | 0.880-0.920 | eGFR ≥60<br>mL/min/1.73 m2<br>(N=1166) | 0.923 | 0.908-0.938 | |
| BMI ≥30.0 kg/m2<br>(N=1160) | 0.903 | 0.885-0.920 | BMI <30.0 kg/m2<br>(N=1025) | 0.945 | 0.931-0.958 | |
| With<br>Comorbidities**<br>(N=1978) | 0.915 | 0.902-0.927 | Without<br>Comorbidities***<br>(N=222) | 0.944 | 0.915-0.974 | |

* Subjects with renal disease on dialysis were excluded from the study

** Subjects with at least one of the following: diabetes, renal insufficiency (eGFR values <60), hypertension (HTN) and/or chronicobstructive pulmonary disease (COPD)

*** Subjects without any of the following: diabetes, renal insufficiency (eGFR values <60), hypertension (HTN) and/or chronic obstructivepulmonary disease (COPD)

Distribution of VITROS NT-proBNP II test results after applying the multiple cutoffs, 300 pg/mL for all age groups and 450pg/mL for 22-<50 years old, 900 pg/mL for 50-<75 years old, 1800 pg/mL for ≥75 years old, versus adjudicated diagnosiswas summarized within and across age groups across gender and is presented in the table below.

# VITROS NT-proBNP II Test Results versus Adjudicated Diagnosis within and across Age Group

| Age<br>(years) | VITROS NT-proBNP II Test Result | Adjudicated Diagnosis | | Total | |
|----------------|---------------------------------------------------|-----------------------|-------------|-------------|-------------|
| | | HF | Non-HF | | |
| 22-<50 | VITROS NT-proBNP II Test<br>Result Interpretation | Positive | 105 | 19 | 124 |
| | | Gray Zone | 5 | 13 | 18 |
| | | Negative | 4 | 109 | 113 |
| | | <b>Total</b> | <b>114</b> | <b>141</b> | <b>255</b> |
| 50-<75 | VITROS NT-proBNP II Test<br>Result Interpretation | Positive | 472 | 115 | 587 |
| | | Gray Zone | 59 | 144 | 203 |
| | | Negative | 7 | 371 | 378 |
| | | <b>Total</b> | <b>538</b> | <b>630</b> | <b>1168</b> |
| ≥75 | VITROS NT-proBNP II Test<br>Result Interpretation | Positive | 372 | 62 | 434 |
| | | Gray Zone | 69 | 153 | 222 |
| | | Negative | 2 | 119 | 121 |
| | | <b>Total</b> | <b>443</b> | <b>334</b> | <b>777</b> |
| All Subjects | VITROS NT-proBNP II Test<br>Result Interpretation | Positive | 949 | 196 | 1145 |
| | | Gray Zone | 133 | 310 | 443 |
| | | Negative | 13 | 599 | 612 |
| | | <b>Total</b> | <b>1095</b> | <b>1105</b> | <b>2200</b> |

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

The following analyses were conducted to determine clinical performance:

| VITROS NT-proBNP II Test<br>Results Classification | Adjudicated Diagnosis | | Total |
|----------------------------------------------------|-----------------------|--------|-------------|
| | HF | Non-HF | |
| Positive: Heart Failure Likely | A | B | A+B |
| Gray Zone: Result<br>Indeterminate | C | D | C+D |
| Negative: Heart Failure<br>Unlikely | E | F | E+F |
| Total | A+C+E | B+D+F | A+B+C+D+E+F |

Pretest Probability of HF (prevalence of HF in the study) = (A+C+E)(A+B+C+D+E+F)

## Posttest Probability of HF

Posttest probability of HF for positive test results = A/(A+B)

Posttest probability of HF for Gray zone test results = C/(C+D)

## Posttest Probability of non-HF

Posttest probability of non-HF for Gray zone test results = D/(C+D)

Posttest probability of non-HF for negative test results = F/(E+F)

## Likelihood ratios (LR) of HF given VITROS NT-proBNP II test result category

LR positive = (A/(A+C+E))/(B/(B+D+F))

LR gray zone = (C/(A+C+E))/(D/(B+D+F))

LR negative = (E/(A+C+E))/(F/(B+D+F))

The pretest probability of HF (prevalence of HF in the study), posttest probabilities, likelihood ratios and the two-tailed 95% CIs of the VITROS NT-proBNP II test result versus adjudicated diagnosis were determined across and within gender using the agedependent rule-in (450 pg/mL for subjects 22-<50 years old; 900 pg/mL for subjects 50-<75 years old: 1800 pg/mLfor subjects >75 years old) and age-independent rule-out (300 pg/mL) cutoffs and are summarized in the following tables:

| Age Group (Years) | Pretest Probability of HF (Prevalence of HF in Study) (n/N) | VITROS NT-proBNP II Test Result Interpretation | Posttest Probability of HF (n/N) | | Posttest Probability of non-HF (n/N) | | Likelihood Ratio Positive (HF) | 95% CI** |
|--------------------|-------------------------------------------------------------|------------------------------------------------|----------------------------------|-------------|--------------------------------------|-------------|--------------------------------|------------|
| | | | Estimate (%) | 95% CI* (%) | Estimate (%) | 95% CI* (%) | | |
| 22-<50<br>(N=255) | 44.7%<br>(114/255) | Positive | 84.7<br>(105/124) | 77.3-90.0 | - | - | 6.84 | 4.48-10.42 |
| | | Gray zone | 27.8<br>(5/18) | 12.5-50.9 | 72.2<br>(13/18) | 49.1-87.5 | 0.48 | 0.17-1.29 |
| | | Negative | - | - | 96.5<br>(109/113) | 91.3-98.6 | 0.05 | 0.02-0.12 |
| 50-<75<br>(N=1168) | 46.1%<br>(538/1168) | Positive | 80.4<br>(472/587) | 77.0-83.4 | - | - | 4.81 | 4.06-5.69 |
| | | Gray zone | 29.1<br>(59/203) | 23.3-35.7 | 70.9<br>(144/203) | 64.3-76.7 | 0.48 | 0.36-0.63 |
| | | Negative | - | - | 98.1<br>(371/378) | 96.2-99.1 | 0.02 | 0.01-0.05 |

### All Subjects

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

| | | | Positive | | | | | |
|-----------------------------|----------------------|-----------|--------------------|-----------|-------------------|-----------|------|-----------|
| ≥75<br>(N=777) | 57.0<br>(443/777) | Positive | 85.7<br>(372/434) | 82.1-88.7 | – | – | 4.52 | 3.60-5.68 |
| | | Gray zone | 31.1<br>(69/222) | 25.4-37.4 | 68.9<br>(153/222) | 62.6-74.6 | 0.34 | 0.27-0.43 |
| | | Negative | – | – | 98.3<br>(119/121) | 94.2-99.5 | 0.01 | 0.00-0.05 |
| All<br>Subjects<br>(N=2200) | 49.8%<br>(1095/2200) | Positive | 82.9<br>(949/1145) | 80.6-85.0 | – | – | 4.89 | 4.29-5.56 |
| | | Gray zone | 30.0<br>(133/443) | 25.9-34.4 | 70.0<br>(310/443) | 65.6-74.1 | 0.43 | 0.36-0.52 |
| | | Negative | – | – | 97.9<br>(599/612) | 96.4-98.8 | 0.02 | 0.01-0.04 |

* 95% Wilson Score Confidence Interval

** Log Method Confidence Interval

The pretest probability of HF (prevalence of HF in the study), posttest probabilities, likelihood ratios and the two-tailed 95%CIs of the VITROS NT-proBNP II test result versus adjudicated diagnosis were determined for the relevant clinical subgroups using the age-dependent rule-in (450 pg/mL for subjects 22-<50 years old; 900 pg/mL for subjects 50-<75 years old; 1800 pg/mL for subjects ≥75 years old) and age-independent rule-out (300 pg/mL) cutoffs and are summarized in the following tables:

| Female Subjects | | | | | | | | |
|----------------------|----------------------------------------------------------------------------|----------------------------------------------------------|-------------------------------------|-----------|-----------------------------------------|-----------|--------------------------------------|------------|
| Age Group<br>(Years) | Pretest<br>Probability<br>of HF<br>(Prevalence<br>of HF in<br>Study) (n/N) | VITROS NT-<br>proBNP II<br>Test Result<br>Interpretation | Posttest Probability of<br>HF (n/N) | | Posttest Probability of<br>non-HF (n/N) | | Likelihood<br>Ratio Positive<br>(HF) | 95% CI** |
| | 37.7%<br>(43/114) | Positive | 88.6<br>(39/44) | 76.0-95.0 | - | - | 12.88 | 5.50-30.15 |
| 22-<50<br>(N=114) | | Gray zone | 11.1<br>(1/9) | 2.0-43.5 | 88.9<br>(8/9) | 56.5-98.0 | 0.21 | 0.03-1.59 |
| | | Negative | - | - | 95.1<br>(58/61) | 86.5-98.3 | 0.09 | 0.03-0.26 |
| | 38.8%<br>(194/500) | Positive | 77.0<br>(167/217) | 70.9-82.1 | - | - | 5.27 | 4.06-6.83 |
| 50-<75<br>(N=500) | | Gray zone | 26.4<br>(23/87) | 18.3-36.6 | 73.6<br>(64/87) | 63.4-81.7 | 0.57 | 0.36-0.88 |
| | | Negative | - | - | 98.0<br>(192/196) | 94.9-99.2 | 0.03 | 0.01-0.09 |
| ≥75<br>(N=402) | 54.0%<br>(217/402) | Positive | 85.9<br>(176/205) | 80.4-90.0 | - | - | 5.17 | 3.68-7.27 |
| | | Gray zone | 30.5<br>(40/131) | 23.3-38.9 | 69.5<br>(91/131) | 61.1-76.7 | 0.37 | 0.27-0.51 |
| | | Negative | - | - | 98.5<br>(65/66) | 91.9-99.7 | 0.01 | 0.00-0.09 |

*95% Wilson Score Confidence Interval

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

| Male Subjects | Age Group<br>(Years) | Pret…