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 Description

The VITROS NT-proBNP II test is performed using the VITROS 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 microwells. The well is incubated for 8 minutes, before unbound materials are removed by washing.

The bound HRP conjugate is measured by a luminescent reaction. A reagent containing luminogenic substrate (a luminol derivative and a peracid salt) and an electron transfer agent, is added to the wells. The HRP in the bound conjugate catalyzes the oxidation of the luminol derivative, producing light. The electron transfer agent (a substituted acetanilide) increases the level of light produced and prolongs its emission. The light signals are read by the System. The amount of HRP conjugate bound is directly proportional to the concentration of NT-proBNP present.

AI/ML Overview

The provided document describes the analytical and clinical performance of the VITROS Immunodiagnostic Products NT-proBNP II Reagent Pack, an in vitro diagnostic device used to aid in the diagnosis and assessment of heart failure.

Here's an analysis of the acceptance criteria and the study proving the device meets them:

1. Table of acceptance criteria and the reported device performance

The document does not explicitly present a "table of acceptance criteria" in terms of pre-defined thresholds for performance metrics that the device must meet for clearance. Instead, it describes the design goals and then reports the observed performance. For clarity, I will create a table summarizing the reported performance, which implicitly indicates the criteria were met or exceeded for FDA clearance.

Test Category	Specific Test / Metric	Acceptance Criteria (Implicit/Design Goal from predicate or general IVD standards)	Reported Device Performance
Analytical Performance
Precision	Repeatability (Within-run) %CV	Typically < 10% for quantitative assays; lower for low concentrations.	Range: 1.0% - 2.1% (SD 0.52 to 370)
	Within-calibration %CV	Typically < 15%	Range: 1.9% - 5.0% (SD 1.63 to 710)
	Within-lab %CV	Typically < 20%	Range: 2.4% - 5.7% (SD 1.91 to 730)
	Total Precision %CV (additional analysis)	Consistent with product performance expectations.	Range: 2.62% - 8.96% (SD 2.86 to 761)
Limit of Detection (LoD)	Designed to be <= 30.0 pg/mL	0.46 pg/mL (Observed); 0.49 pg/mL (Claimed)
Limit of Quantitation (LoQ)	Designed to be <= 30.0 pg/mL at 20% CV	0.46 pg/mL at 20% CV (Observed); 20.0 pg/mL (Claimed, to maintain linearity)
Linearity	Across measuring range	Linear over the measuring range	Linear from 20.0 to 30,000 pg/mL
Matrix Comparison	Serum vs. EDTA plasma vs. Lithium Heparin plasma	No significant effect, meet acceptance criteria.	All three tube types suitable for use (met acceptance criteria).
Analytical Specificity	Bias from common substances < 10%	No bias > 10% observed for most tested compounds.	Specific interferents (Cefoxitin sodium, Sodium Azide) showed >10% bias.
Cross-Reactivity	Various related peptides (e.g., ANP, proBNP, BNP32)	Low cross-reactivity desired.	Ranges from <1.0% to 39.1% (non-glycosylated proBNP). Most are <1%.
High Dose Hook Effect	No effect up to X concentration	No high dose hook effect up to 300,000 pg/mL.
Clinical Performance
Aid in HF Diagnosis (ED Setting)	AUC (Overall)	Not explicitly stated, but typically high (e.g., > 0.85 or 0.90 for this type of test)	0.920 (95% CI: 0.909-0.931)
	AUC (Age-stratified)		Ranged from 0.904 to 0.954
	AUC (Clinical Subgroups)		Ranged from 0.899 to 0.945
	Posttest Probability of HF (Positive result)	High positive predictive value/posttest probability for rule-in.	Range: 80.4% - 85.7% across age groups
	Posttest Probability of non-HF (Negative result)	High negative predictive value/posttest probability for rule-out.	Range: 96.5% - 98.3% across age groups
	Likelihood Ratio Positive (LR+)	High (e.g., > 5-10 for strong rule-in)	Range: 4.52 - 6.84 across age groups
	Likelihood Ratio Negative (LR-)	Low (e.g., < 0.1-0.2 for strong rule-out)	Range: 0.01 - 0.05 across age groups
Aid in HF Diagnosis (Outpatient Setting)	AUC (Overall)		0.880 (95% CI: 0.822 to 0.937)
	AUC (Clinical Subgroups)		Ranged from 0.838 to 0.940
	Sensitivity (Rule-out cutoff 125 pg/mL)	High (for rule-out, e.g., >90%)	91.7% (44/48)
	Specificity (Rule-out cutoff 125 pg/mL)	Reasonable (for rule-out, may be lower)	67.2% (490/729)
	NPV (Rule-out cutoff 125 pg/mL)	High (for rule-out, e.g., >90%)	99.2% (490/494)
	PPV (Rule-out cutoff 125 pg/mL)	(for rule-out, may be lower)	15.6% (44/283)
Correlation with NYHA	Statistical significance of relationship with HF severity	Statistically significant trend.	Jonckheere-Terpstra test p < 0.0001 (statistically significant correlation).

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

ED Setting (Diagnosis of Heart Failure):
- Sample Size: 2200 subjects.
- Data Provenance: Multi-center prospective study, 20 collection sites across the United States.
Outpatient Setting (Diagnosis of Heart Failure):
- Sample Size: 777 subjects.
- Data Provenance: Multi-center prospective study, 10 collection sites across the United States.
Correlation with NYHA Functional Classification:
- Sample Size: 1143 subjects with heart failure.
- Data Provenance: Not explicitly stated if this was a separate collection or a subset of the ED/outpatient studies, but given it's "subjects with heart failure," it likely derives from similar clinical populations.

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

For both the ED and Outpatient settings: The final clinical diagnosis (ground truth) was adjudicated by independent cardiologists or ED physicians experienced in diagnosing HF.
Number of Experts: Not specified. The document only mentions "independent cardiologists or ED physicians," implying more than one, but not a precise number.
Qualifications: "Experienced in diagnosing HF." No specific number of years of experience or board certifications are mentioned.

4. Adjudication method (e.g. 2+1, 3+1, none) for the test set

The document states that the final clinical diagnosis was "adjudicated by independent cardiologists or ED physicians experienced in diagnosing HF." It does not specify a quantitative adjudication method like "2+1" or "3+1." This suggests that the adjudication process was qualitative and relied on the clinical expertise of the adjudicators to reach a consensus diagnosis for each patient, rather than a fixed number of readers and a tie-breaking rule.

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, a MRMC comparative effectiveness study was not done. This document describes the performance validation of an in vitro diagnostic (IVD) immunoassay kit (VITROS NT-proBNP II Reagent Pack), which measures blood biomarkers. This is not an AI/imaging device where human readers would typically be assisted by AI. The product is a laboratory test, and its performance is evaluated based on its accuracy in measuring the biomarker and its diagnostic utility compared to clinical truth.

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

Yes, this is effectively a standalone performance study. The VITROS NT-proBNP II Reagent Pack provides a quantitative measurement of NT-proBNP. Its performance is assessed purely on the analytical accuracy of the measurement and its direct correlation with clinical outcomes (diagnosis of HF, NYHA classification) as determined by the expert adjudicators, without human interpretation of the device's numerical output for diagnosis (i.e., it's a direct measurement, not an interpretative AI). The output values are then interpreted against established cutoffs to aid in diagnosis, but the device itself does not involve a human-in-the-loop for its direct performance.

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

The primary ground truth for heart failure diagnosis was expert clinical judgment/adjudication. For the diagnostic studies, "The final clinical diagnosis was adjudicated by independent cardiologists or ED physicians experienced in diagnosing HF."
For the assessment of heart failure severity, the ground truth was New York Heart Association (NYHA) Functional Classification, which is a clinical classification based on patient symptoms and physical activity levels.

8. The sample size for the training set

This document describes the validation of an in vitro diagnostic device (a reagent pack and system) for measuring a biomarker, not a machine learning or AI algorithm in the typical sense that would involve a "training set" for model development. The assays (immunoassays) are based on chemical reactions rather than statistical models trained on large datasets.
The "training" of such a device primarily involves rigorous analytical development and characterization, ensuring the chemical processes are precise and accurate. Therefore, the concept of a separate "training set" with ground truth data, as used in AI/ML, is not directly applicable here. The document focuses on the performance validation of the developed assay.

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

As noted above, the concept of a "training set" with ground truth for an AI/ML algorithm doesn't directly apply to this type of IVD immunoassay. The analytical methods and performance characteristics (precision, linearity, LoD, LoQ, analytical specificity, etc.) are established through laboratory experiments and characterization studies, not by training on a clinical dataset with ground truth in the AI/ML sense. Clinical performance is then validated against independently established clinical diagnoses (expert adjudication).

Summary

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

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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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: (585) 453-4152 F: (585) 453-3113 Contact Person: Ann Quinn, Director, Regulatory Affairs
September 23, 2021 2. Preparation Date
1. 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.

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	microwells. The well is incubated for 8 minutes, before unboundmaterials are removed by washing.
	The bound HRP conjugate is measured by a luminescent reaction. Areagent containing luminogenic substrate (a luminol derivative and aperacid salt) and an electron transfer agent, is added to the wells. The HRPin the bound conjugate catalyzes the oxidation of the luminol derivative,producing light. The electron transfer agent (a substituted acetanilide)increases the level of light produced and prolongs its emission. The lightsignals are read by the System. The amount of HRP conjugate bound isdirectly proportional to the concentration of NT-proBNP present.
6. Deviceintendeduse	Rx ONLYFor in vitro diagnostic use only.
	For the quantitative measurement of N-terminal pro Brain NatriureticPeptide (NT-proBNP) in human serum and plasma (K2 EDTA or LithiumHeparin) using the VITROS 3600 Immunodiagnostic System to aid in thediagnosis of heart failure. The test can also be used in the assessment ofheart failure severity in patients diagnosed with heart failure.

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

DeviceCharacteristic	New DeviceVITROS NT-proBNP II	Predicate DeviceRoche Elecsys proBNPII Immunoassay,K072437
Intended Use	Rx ONLYFor in vitro diagnostic use only.For the quantitative measurement of N-terminal pro BrainNatriuretic Peptide (NT-proBNP) in human serum andplasma (K2 EDTA or Lithium Heparin) using the VITROS3600 Immunodiagnostic System to aid in the diagnosis ofheart failure. The test can also be used in the assessment ofheart failure severity in patients diagnosed with heartfailure.	For the quantitativedetermination of N-terminal pro-Brainnatriuretic peptide inhuman serum andplasma. Elecsys proBNPII assay is used as an aidin the diagnosis ofindividuals suspected ofhaving congestive heartfailure. The test isfurther indicated for therisk stratification ofpatients with acutecoronary syndrome andcongestive heart failure.The test may also serve
DeviceCharacteristic	New DeviceVITROS NT-proBNP II	Predicate DeviceRoche Elecsys proBNPII Immunoassay,K072437
Intended Use	See above	as an aid in theassessment of increasedrisk of cardiovascularevents and mortality inpatients at risk for heartfailure who have stablecoronary artery disease.The electro-chemiluminescenseimmunoassay "ECLIA"is intended for use onElecsys and cobas eimmunoassay analyzers.
Basic Principle	Sandwich immunoassay	Same
Antibody	Monoclonal anti-NT-proBNP	Same
Sample Type	Serum and plasma	Same
MeasuringRange	20-30,000 pg/mL	5-35,000 pg/mL
Traceability	Standardized against the Elecsys proBNP II assay.	Standardized against theElecsys proBNP assay.<75 years: 125 pg/mL≥75 years: 450 pg/mL
ResultInterpretation	For patients presenting to the ED settings with acute orworsening dyspnea and clinical suspicion of HF, theVITROS NT-proBNP II test results should be interpreted asindicated in the table below.
	VITROS NT-proBNP II Test Results(pg/mL)	Age Group (Years)	Interpretation of Results
	<300	All	Negative: Heart Failure Unlikely
	≥300 to <450	22-<50	Gray Zone: Result Indeterminate -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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DeviceCharacteristic	New DeviceVITROS NT-proBNP II	Predicate DeviceRoche Elecsys proBNPII Immunoassay,K072437
	For ambulatory patients presenting to outpatient facilitieswith clinical suspicion of HF not previously diagnosed andat least one sign, symptom or risk factor for HF, theVITROS NT-proBNP II test results should be interpreted asindicated in the table below.
	VITROS NT-proBNP II Test Results(pg/mL) Age Group Interpretation of Results <125 All Negative: Heart Failure Unlikely ≥125 All Consider Heart Failure as well as othercauses* 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.Observations	No.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-proBNPConc.	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	Meanpg/mL	Between Lot*		Between Day**		BetweenRuns***		Within Run /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
Atorvastatincalciumtrihydrate	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
Ceftriaxonedisodiumhemi(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
Clopidogrelhydrogensulfate	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	Phenprocoumon(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-methylaminoantipyrineHydrochloride)	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(Nitroglycerin)	1.20 µg/dL	52.8 nmol/L	Valproic Acid	31.8 mg/dL	2.21 mmol/L
HAMA (Human Anti-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.

{10}------------------------------------------------

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(preproANP25-55)	3.50 µg/mL	0.998 µmol/L	*	*	*	*	*
NT-proANP31-67(preproANP56-92)	1.00 ng/mL	0.258 nmol/L	*	*	*	*	*
NT-proANP79-98(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-proBNPResult of ControlSample		Mean NT-proBNPResult of Cross-Reactant Sample		% Cross-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

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NT-proANP1-30(preproANP25-55)	3.50 µg/mL	0.998 µmol/L	113	13.3	775	91.5	<1.0
NT-proANP31-67(preproANP56-92)	1.00 ng/mL	0.258 nmol/L	112	13.2	113	13.3	<1.0
NT-proANP79-98(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 LowerLimit(pg/mL)	RI UpperLimit(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

StudyPopulation	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

StudyPopulation	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

StudyPopulation	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% ConfidenceInterval	AUC	95% ConfidenceInterval	AUC	95% ConfidenceInterval
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(N=1220)	0.899	0.881-0.918	No History of HF(N=970)	0.932	0.917-0.947
eGFR <60*mL/min/1.73 m2(N=1012)	0.900	0.880-0.920	eGFR ≥60mL/min/1.73 m2(N=1166)	0.923	0.908-0.938
BMI ≥30.0 kg/m2(N=1160)	0.903	0.885-0.920	BMI <30.0 kg/m2(N=1025)	0.945	0.931-0.958
WithComorbidities**(N=1978)	0.915	0.902-0.927	WithoutComorbidities***(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(years)	VITROS NT-proBNP II Test Result	Adjudicated Diagnosis		Total
		HF	Non-HF
22-<50	VITROS NT-proBNP II TestResult Interpretation	Positive	105	19	124
		Gray Zone	5	13	18
		Negative	4	109	113
		Total	114	141	255
50-<75	VITROS NT-proBNP II TestResult Interpretation	Positive	472	115	587
		Gray Zone	59	144	203
		Negative	7	371	378
		Total	538	630	1168
≥75	VITROS NT-proBNP II TestResult Interpretation	Positive	372	62	434
		Gray Zone	69	153	222
		Negative	2	119	121
		Total	443	334	777
All Subjects	VITROS NT-proBNP II TestResult Interpretation	Positive	949	196	1145
		Gray Zone	133	310	443
		Negative	13	599	612
		Total	1095	1105	2200

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

The following analyses were conducted to determine clinical performance:

VITROS NT-proBNP II TestResults Classification	Adjudicated Diagnosis		Total
	HF	Non-HF
Positive: Heart Failure Likely	A	B	A+B
Gray Zone: ResultIndeterminate	C	D	C+D
Negative: Heart FailureUnlikely	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(N=255)	44.7%(114/255)	Positive	84.7(105/124)	77.3-90.0	-	-	6.84	4.48-10.42
		Gray zone	27.8(5/18)	12.5-50.9	72.2(13/18)	49.1-87.5	0.48	0.17-1.29
		Negative	-	-	96.5(109/113)	91.3-98.6	0.05	0.02-0.12
50-<75(N=1168)	46.1%(538/1168)	Positive	80.4(472/587)	77.0-83.4	-	-	4.81	4.06-5.69
		Gray zone	29.1(59/203)	23.3-35.7	70.9(144/203)	64.3-76.7	0.48	0.36-0.63
		Negative	-	-	98.1(371/378)	96.2-99.1	0.02	0.01-0.05

All Subjects

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

			Positive
≥75(N=777)	57.0(443/777)	Positive	85.7(372/434)	82.1-88.7	–	–	4.52	3.60-5.68
		Gray zone	31.1(69/222)	25.4-37.4	68.9(153/222)	62.6-74.6	0.34	0.27-0.43
		Negative	–	–	98.3(119/121)	94.2-99.5	0.01	0.00-0.05
AllSubjects(N=2200)	49.8%(1095/2200)	Positive	82.9(949/1145)	80.6-85.0	–	–	4.89	4.29-5.56
		Gray zone	30.0(133/443)	25.9-34.4	70.0(310/443)	65.6-74.1	0.43	0.36-0.52
		Negative	–	–	97.9(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(Years)	PretestProbabilityof HF(Prevalenceof HF inStudy) (n/N)	VITROS NT-proBNP IITest ResultInterpretation	Posttest Probability ofHF (n/N)		Posttest Probability ofnon-HF (n/N)		LikelihoodRatio Positive(HF)	95% CI**
	37.7%(43/114)	Positive	88.6(39/44)	76.0-95.0	-	-	12.88	5.50-30.15
22-<50(N=114)		Gray zone	11.1(1/9)	2.0-43.5	88.9(8/9)	56.5-98.0	0.21	0.03-1.59
		Negative	-	-	95.1(58/61)	86.5-98.3	0.09	0.03-0.26
	38.8%(194/500)	Positive	77.0(167/217)	70.9-82.1	-	-	5.27	4.06-6.83
50-<75(N=500)		Gray zone	26.4(23/87)	18.3-36.6	73.6(64/87)	63.4-81.7	0.57	0.36-0.88
		Negative	-	-	98.0(192/196)	94.9-99.2	0.03	0.01-0.09
≥75(N=402)	54.0%(217/402)	Positive	85.9(176/205)	80.4-90.0	-	-	5.17	3.68-7.27
		Gray zone	30.5(40/131)	23.3-38.9	69.5(91/131)	61.1-76.7	0.37	0.27-0.51
		Negative	-	-	98.5(65/66)	91.9-99.7	0.01	0.00-0.09