Predicate Devices

Reference Devices

Not Found

AI/ML (Artificial Intelligence / Machine Learning)

No
The device description and performance studies focus on standard immunoassay technology and statistical analysis of results compared to a cutoff value and a predicate device. There is no mention of AI, ML, or related concepts.

Therapeutic

No
Explanation: This device is an in vitro diagnostic (IVD) immunoassay designed for detecting antibodies to hepatitis A virus, which aids in diagnosis and identifying susceptibility or vaccine response. It does not provide any form of therapy or treatment.

Diagnostic

Yes

The device "Elecsys Anti-HAV II" is explicitly indicated as "an aid in the clinical laboratory diagnosis of acute or past hepatitis A virus infection" and "an aid to identify HAV susceptible individuals and to determine the presence of an antibody response to HAV in vaccine recipients," which are direct diagnostic purposes.

SaMD (Software as a Medical Device)

No

The device is an immunoassay that requires reagents and a specific hardware analyzer (cobas e immunoassay analyzers) to function. While software is used for result determination, it is an integral part of a larger hardware/reagent system, not a standalone software-only device.

IVD (In Vitro Diagnostic)

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

Here's why:

Intended Use: The "Intended Use / Indications for Use" section explicitly states "Immunoassay for the in vitro qualitative detection of total antibodies (IgG and IgM) to hepatitis A virus (HAV) in human pediatric... and adult serum and plasma". The phrase "in vitro" is a key indicator of an IVD.
Purpose: The assay is intended as an aid in the clinical laboratory diagnosis of acute or past hepatitis A virus infection, to identify susceptible individuals, and to determine antibody response to vaccination. These are all diagnostic purposes performed on samples taken from the body.
Sample Type: The assay uses human serum and plasma, which are biological specimens taken from the body.
Device Description: The description details a laboratory-based immunoassay system (Elecsys Anti-HAV II on cobas e analyzers) that analyzes these biological samples to produce a result.
Performance Studies: The document describes performance studies conducted on human serum and plasma samples to evaluate the device's analytical and clinical performance.

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

PCCP (Predetermined Change Control Plan) Authorized

N/A

Overview

Intended Use / Indications for Use

Immunoassay for the in vitro qualitative detection of total antibodies (IgG and IgM) to hepatitis A virus (HAV) in human pediatric (ages 2 through 21 years) and adult serum and plasma (Li-Heparin, potassium EDTA, Na-Citrate, Na-Heparin). The assay, in conjunction with other serological and clinical information, is indicated as an aid in the clinical laboratory diagnosis of acute or past hepatitis A virus infection in persons with signs or symptoms of hepatitis and in persons at increased risk for hepatitis A infection, or as an aid to identify HAV susceptible individuals and to determine the presence of an antibody response to HAV in vaccine recipients.

The electrochemiluminescence immunoassay "ECLIA" is intended for use on the cobas e immunoassay analyzers.

Assay performance characteristics have not been established for immunocompromised or immunosuppressed patients. This assay has not been FDA cleared or approved for the screening of blood or plasma donors.

Product codes (comma separated list FDA assigned to the subject device)

LOL, QCH

Device Description

Elecsys Anti-HAV II is a second generation assay by Roche Diagnostics for the in vitro qualitative detection of total antibodies (IgG and IgM) to the hepatitis A virus (HAV) in human pediatric (ages 2 through 21 years) and adult serum and plasma. It is intended for use on the cobas e 601 immunoassay analyzer. The cobas e family of analyzers employs the electrochemiluminescence "ECLIA" technology. The assay is an 18-minute assay utilizing a competition principle.

Results are determined automatically by the software by comparing the electrochemiluminescence signal obtained from the reaction product of the sample with the signal of the cutoff value previously obtained by calibration.

The reagent rackpack working solutions include:

M: Streptavidin-coated microparticles .
R1: HAV Ag (cell culture) .
R2: Biotinylated monoclonal anti-HAV antibody, monoclonal Anti-HAV antibody . labeled with ruthenium complex
AHAV 2 Cal1: Negative Calibrator 1 (human serum) .
AHAV 2 Cal2: Positive Calibrator 2 (anti-HAV (human), approximately 60 IU/L in . human serum)

PreciControl Anti-HAV II is a ready-for-use control serum based on human serum both in the negative and positive concentration range. The controls are used for monitoring the performance of the Elecsys Anti-HAV II immunoassay. PreciControl Anti-HAV II is sold separately from the Elecsys Anti-HAV II immunoassay reagent.

Mentions image processing

Not Found

Mentions AI, DNN, or ML

Not Found

Input Imaging Modality

Not Found

Anatomical Site

Not Found

Indicated Patient Age Range

pediatric (ages 2 through 21 years) and adult

Intended User / Care Setting

clinical laboratory

Description of the training set, sample size, data source, and annotation protocol

Not Found

Description of the test set, sample size, data source, and annotation protocol

Not Found

Summary of Performance Studies (study type, sample size, AUC, MRMC, standalone performance, key results)

Precision (Repeatability and Within-Laboratory Precision):

Study type: Repeatability and Within-Laboratory Precision based on CLSI EP05-A3.
Sample size: 84 per sample type (2 aliquots each of two levels of control and 5 human sera per run, 2 runs per day for 21 days).
Key results:
- Human serum 1: Mean (COI) 1.42, Repeatability SD 0.016, CV 1.1%; Within-Laboratory SD 0.028, CV 2.0%.
- Human serum 2: Mean (COI) 1.15, Repeatability SD 0.012, CV 1.1%; Within-Laboratory SD 0.022, CV 1.9%.
- Human serum 3: Mean (COI) 0.955, Repeatability SD 0.009, CV 0.9%; Within-Laboratory SD 0.022, CV 2.3%.
- Human serum 4: Mean (COI) 0.665, Repeatability SD 0.009, CV 1.3%; Within-Laboratory SD 0.020, CV 2.9%.
- Human serum 5: Mean (COI) 0.006, Repeatability SD 0.0002, CV 3.0%; Within-Laboratory SD 0.0002, CV 3.3%.
- PC Anti-HAV II 1: Mean (COI) 1.30, Repeatability SD 0.015, CV 1.1%; Within-Laboratory SD 0.024, CV 1.8%.
- PC Anti-HAV II 2: Mean (COI) 0.339, Repeatability SD 0.005, CV 1.5%; Within-Laboratory SD 0.009, CV 2.7%.

Analytical Sensitivity (Determination of Cut-off Sensitivity):

Study type: Internal studies and external clinical studies using serial dilutions of the 2nd International Standard for Anti-Hepatitis A, Immunoglobulin, Human, NIBSC code: 97/646.
Sample size: 10 serially diluted samples, tested fourfold with three different reagent and calibrator lots.
Key results: The cut-off of COI = 1.0 corresponds to ≤ 25.4 IU/L.
- Lot MP01: Cut-off sensitivity 24.6 IU/L.
- Lot PoQ: Cut-off sensitivity 25.4 IU/L.
- Lot P02: Cut-off sensitivity 20.1 IU/L.

Human Anti-Mouse Antibodies (HAMA):

Study type: Evaluation of HAMA interference.
Sample size: 11 human serum samples.
Key results: No HAMA interference was found within predefined acceptance criteria.

Endogenous Interferences (Hemoglobin/Bilirubin/Lipemia, Rheumatoid Factor, Serum Albumin, IgG, IgA, and IgM):

Study type: Evaluation of endogenous substances for potential interference in accordance with CLSI EP07-A2.
Sample size: Four human serum samples for each interfering substance.
Key results: All results met pre-defined acceptance criteria, demonstrating no interference from endogenous substances up to the levels: Intralipid® (Lipemia) 2000 mg/dL, Bilirubin 66 mg/dL, Hemoglobin 1000 mg/dL, Rheumatoid Factor 1400 IU/mL, human Serum albumin 7.00 g/dL, human IgG 7.00 g/dL, human IgM 1.00 g/dL, human IgA 1.60 g/dL.

Biotin Interference:

Study type: Evaluation of biotin interference in accordance with CLSI EP07-A2.
Sample size: Five human serum samples.
Key results: Negative specimens with biotin concentrations up to 100 ng/mL demonstrated ≤11% negative bias. Biotin concentrations greater than 100 ng/mL can lead to higher negative bias and false positive results.

Analytical Specificity/Cross-Reactivity:

Study type: Evaluation of potential cross-reacting antibodies.
Sample size: 10 samples each for acute Hepatitis B, acute Hepatitis C, HIV, EBV, Anti-CMV, Anti-HSV, Toxoplasma Gondii, Treponema Pallidum, Anti-Mumps/Rubeola, Anti-Rubella, Anti-Parvovirus B19, and ANA Autoimmune antibodies.
Key results: No cross-reactivity with other infectious agents was found.

Exogenous Interferences – Drugs:

Study type: Evaluation of interference from 18 commonly used pharmaceutical compounds in accordance with CLSI EP07-A2.
Sample size: Four human serum samples.
Key results: All results met pre-defined acceptance criteria, demonstrating no interference from the drug substances at the tested concentrations.

Sample Matrix Comparison:

Study type: Comparison of results from serum and plasma collection tubes.
Sample size: At least 60 serum/plasma pairs for each anticoagulant (K2-EDTA, K3-EDTA, Na-Heparin, Li-Heparin, Na-Citrate).
Key results: Specifications were met for all anticoagulants, demonstrating acceptability of the sample types.

Analytical Method Comparison to Predicate:

Study type: Method comparison versus Elecsys Anti-HAV (K100903) conducted according to CLSI EP09-A3.
Sample size: 215 modified serum samples (≥100 negative and ≥100 positive samples).
Key results:
- Positive percent agreement rate: 100% (107/107).
- Negative percent agreement rate: 100% (98/98).
- 20% (2/10) of borderline predicate samples were positive, and 80% (8/10) were negative by the Elecsys Anti-HAV II.
- Results met pre-defined acceptance criteria, demonstrating equivalency.

Reagent Stability:

Study type: Reagent stability after first opening (8 weeks) and on-board reagent stability (8 weeks).
Key results: All pre-defined acceptance criteria were met for both studies, demonstrating stability.

Calibration Stability:

Study type: Lot calibration stability (4 weeks) and on-board calibration stability (1 week).
Key results: All pre-defined acceptance criteria were met for both studies.

Sample Stability:

Study type: Sample stability at 2-8°C (14 days), room temperature (15-25°C) (6 days), -15 to -25°C (3 months), and freeze/thaw cycles (5 cycles).
Sample size: Six or seven human serum/plasma samples for each study.
Key results: All pre-defined acceptance criteria were met, demonstrating stability for each condition.

Reproducibility:

Study type: Precision study across three external testing sites using three different cobas e 601 immunoassay analyzers, based on CLSI EP05-A3.
Sample size: 90 replicates for each of four human serum pools and two PreciControl materials.
Key results: Reproducibility %CV ranged from 2.2% to 4.8%.

Method Comparison Versus Predicate (Clinical):

Study type: Comparison of Elecsys Anti-HAV II to Elecsys Anti-HAV (predicate) at three clinical laboratories.
Sample size: 961 specimens from various cohorts.
Key results:
- Overall PPA: 99.80% (501/502), 95% CI (98.90, 99.99).
- Overall NPA: 95.21% (437/459), 95% CI (92.83, 96.97).
- The lower end of the 95% confidence intervals for PPA ranged from 90.45% (hospitalized cohort) to 97.18% (symptomatic cohort).
- The lower end of the ≥ 95% confidence intervals for NPA ranged from 52.36% (pediatric cohort) to 93.04% (hospitalized cohort).
- Overall percentages for all cohorts combined met the expected performance of a lower bound of the 95% confidence interval of ≥ 90% for the agreement rates.

Pre- and Post-HAV Vaccination:

Study type: Evaluation of specimens collected pre- and post-HAV vaccination.
Sample size: 49 subjects.
Key results: No discrepant results were observed.

Prevalence Study:

Study type: Evaluation of HAV antibodies prevalence in apparently healthy population.
Sample size: 400 evaluable subjects from Western US and 400 evaluable subjects from Eastern US.
Key results: Results consistent with higher prevalence in Western US (53.75%) versus Eastern US (29.50%).

Key Metrics (Sensitivity, Specificity, PPV, NPV, etc.)

Analytical Sensitivity (Cut-off Sensitivity):

Cut-off of COI = 1.0 corresponds to ≤ 25.4 IU/L. Specific examples for different lots: 24.6 IU/L, 25.4 IU/L, 20.1 IU/L.

Analytical Method Comparison to Predicate:

Positive percent agreement (PPA): 100% (107/107)
Negative percent agreement (NPA): 100% (98/98)

Clinical Method Comparison Versus Predicate (Overall All Sites Combined):

PPA: 99.80% (501/502), 95% CI (98.90; 99.99)
NPA: 95.21% (437/459), 95% CI (92.83; 96.97)

Clinical Method Comparison Versus Predicate (Individual Cohorts):

Routine HAV testing: PPA 100 (91/91) (96.03, 100); NPA 94.50 (103/109) (88.40, 97.95)
Hospitalized: PPA 98.21 (55/56) (90.45, 99.95); NPA 97.22 (140/144) (93.04, 99.24)
Increased risk for hepatitis: PPA 100 (119/119) (96.95, 100); NPA 94.25 (82/87) (87.10, 98.11)
Symptomatic: PPA 100 (129/129) (97.18, 100); NPA 96.70 (88/91) (90.67, 99.31)
Characterized acute HAV: PPA 100 (65/65) (94.48, 100); NPA 100 (10/10) (69.15, 100)
Pediatric: PPA 100 (42/42) (91.59, 100); NPA 77.78 (14/18) (52.36, 93.59)

Predicate Device(s): If the device was cleared using the 510(k) pathway, identify the Predicate Device(s) K/DEN number used to claim substantial equivalence and list them here in a comma separated list exactly as they appear in the text. List the primary predicate first in the list.

Elecsys Anti-HAV (K100903)

Reference Device(s): Identify the Reference Device(s) K/DEN number and list them here in a comma separated list exactly as they appear in the text.

Not Found

Predetermined Change Control Plan (PCCP) - All Relevant Information for the subject device only (e.g. presence / absence, what scope was granted / cleared under the PCCP, any restrictions, etc).

Not Found

Regulation Number and Section

§ 866.3310 Hepatitis A virus (HAV) serological assays.

(a)
Identification. HAV serological assays are devices that consist of antigens and antisera for the detection of hepatitis A virus-specific IgM, IgG, or total antibodies (IgM and IgG), in human serum or plasma. These devices are used for testing specimens from individuals who have signs and symptoms consistent with acute hepatitis to determine if an individual has been previously infected with HAV, or as an aid to identify HAV-susceptible individuals. The detection of these antibodies aids in the clinical laboratory diagnosis of an acute or past infection by HAV in conjunction with other clinical laboratory findings. These devices are not intended for screening blood or solid or soft tissue donors.(b)
Classification. Class II (special controls). The special control is “Guidance for Industry and FDA Staff: Class II Special Controls Guidance Document: Hepatitis A Virus Serological Assays.” See § 866.1(e) for the availability of this guidance document.

Summary

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Image /page/0/Picture/0 description: The image shows 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 square are the words "U.S. FOOD & DRUG" in blue, with the word "ADMINISTRATION" below it.

August 13, 2019

Roche Diagnostics Jamie Ferguson Regulatory Affairs Principal 9115 Hague Road Indianapolis, Indiana 46250

Re: K190428

Trade/Device Name: Elecsys Anti-HAV II Regulation Number: 21 CFR 866.3310 Regulation Name: Hepatitis A Virus (HAV) Serological Assays Regulatory Class: Class II Product Code: LOL, QCH Dated: February 20, 2019 Received: February 22, 2019

Dear Jamie Ferguson:

We have reviewed your Section 510(k) premarket notification of intent to market the device referenced above and have determined the device is substantially equivalent (for the indications for use stated in the enclosure) to legally marketed predicate devices marketed in interstate commerce prior to May 28, 1976, the enactment date of the Medical Device Amendments, or to devices that have been reclassified in accordance with the provisions of the Federal Food, Drug, and Cosmetic Act (Act) that do not require approval of a premarket approval application (PMA). You may, therefore, market the device, subject to the general controls provisions of the Act. Although this letter refers to your product as a device, please be aware that some cleared products may instead be combination products. The 510(k) Premarket Notification Database located at https://www.accessdata.fda.gov/scripts/cdrh/cfdocs/cfpmn/pmn.cfm identifies combination product submissions. The general controls provisions of the Act include requirements for annual registration, listing of devices, good manufacturing practice, labeling, and prohibitions against misbranding and adulteration. Please note: CDRH does not evaluate information related to contract liability warranties. We remind you, however, that device labeling must be truthful and not misleading.

If your device is classified (see above) into either class II (Special Controls) or class III (PMA), it may be subject to additional controls. Existing major regulations affecting your device can be found in the Code of Federal Regulations, Title 21, Parts 800 to 898. In addition, FDA may publish further announcements concerning your device in the Federal Register.

Please be advised that FDA's issuance of a substantial equivalence determination does not mean that FDA has made a determination that your device complies with other requirements of the Act or any Federal statutes and regulations administered by other Federal agencies. You must comply with all the Act's

1

requirements, including, but not limited to: registration and listing (21 CFR Part 807); labeling (21 CFR Part 801 and Part 809); medical device reporting of medical device-related adverse events) (21 CFR 803) for devices or postmarketing safety reporting (21 CFR 4, Subpart B) for combination products (see https://www.fda.gov/combination-products/guidance-regulatory-information/postmarketing-safety-reportingcombination-products); good manufacturing practice requirements as set forth in the quality systems (OS) regulation (21 CFR Part 820) for devices or current good manufacturing practices (21 CFR 4, Subpart A) for combination products; and, if applicable, the electronic product radiation control provisions (Sections 531-542 of the Act); 21 CFR 1000-1050.

Also, please note the regulation entitled, "Misbranding by reference to premarket notification" (21 CFR Part 807.97). For questions regarding the reporting of adverse events under the MDR regulation (21 CFR Part 803), please go to https://www.fda.gov/medical-device-safety/medical-device-reportingmdr-how-report-medical-device-problems.

For comprehensive regulatory information about mediation-emitting products, including information about labeling regulations, please see Device Advice (https://www.fda.gov/medicaldevices/device-advice-comprehensive-regulatory-assistance) and CDRH Learn (https://www.fda.gov/training-and-continuing-education/cdrh-learn). Additionally, you may contact the Division of Industry and Consumer Education (DICE) to ask a question about a specific regulatory topic. See the DICE website (https://www.fda.gov/medical-device-advice-comprehensive-regulatoryassistance/contact-us-division-industry-and-consumer-education-dice) for more information or contact DICE by email (DICE@fda.hhs.gov) or phone (1-800-638-2041 or 301-796-7100).

Sincerely,

for

Maria Garcia, Ph.D. Chief Division of Microbiology Devices OHT7: Office of In Vitro Diagnostics and Radiological Health Office of Product Evaluation and Quality Center for Devices and Radiological Health

Enclosure

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Indications for Use

510(k) Number (if known) K190428

Device Name Elecsys Anti-HAV II

Indications for Use (Describe)

Immunoassay for the in vitro qualitative detection of total antibodies (IgG and IgM) to hepatitis A virus (HAV) in human pediatric (ages 2 through 21 years) and adult serum and plasma (Li-heparin, potassium EDTA, Na-hebarin). The assay, in conjunction with other serological and clinical information, is indicated as an aid in the clinical laboratory diagnosis of acute or past hepatitis A virus infection in persons with signs or symptoms of hepatitis and in persons at increased risk for hepatitis A infection, or as an aid to identify HAV susceptible individuals and to determine the presence of an antibody response to HAV in vaccine recipients.

The electrochemiluminescence immunoassay "ECLIA" is intended for use on the cobas e immunoassay analyzers.

Assay performance characteristics have not been established for immunocompromised or immunosuppressed patients. This assay has not been FDA cleared or approved for the screening of blood or plasma donors.

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

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Elecsys Anti-HAV II 510(k) Summary

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

In accordance with 21 CFR 807.87, Roche Diagnostics hereby submits official notification as required by Section 510(k) of the Federal Food, Drug and Cosmetics Act of our intention to market the device described in this Premarket Notification 510(k).

The purpose of this Traditional 510(k) Premarket Notification is to obtain FDA review and clearance for the Elecsys Anti-HAV II.

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Submitter Name	Roche Diagnostics
Address	9115 Hague Road
P.O. Box 50416
Indianapolis, IN 46250-0457
Contact	Jamie Ferguson
Phone: (317) 521-4213
Fax: (317) 521-2324
Email: Jamie.ferguson@roche.com
	Tammy Dean
Phone: (317) 521-3978
Fax: (317) 521-2324
Email: tammy.dean@roche.com
Date Prepared	February 20, 2019
Proprietary Name	Elecsys Anti-HAV II
PreciControl Anti-HAV II
Common Name	Anti-HAV II
PreciControl Anti-HAV II
Classification Name	1. Hepatitis A virus (HAV) Serological Assays

Assayed Quality Control Material for Clinical Microbiology Assays |
| Product Codes,
Regulation Numbers | 1. LOL, 21 CFR §866.3310
QCH, 21 CFR §866.3920, Quality Control |
| Predicate Devices | Elecsys Anti-HAV (K100903) |
| Establishment Registration | For the Elecsys Anti-HAV II, the establishment registration number for Roche
Diagnostics GmbH in Mannheim, Germany is 9610126, and for Penzberg,
Germany, 9610529. The establishment registration number for Roche
Diagnostics in the United States is 1823260. |

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DEVICE DESCRIPTION 1.

Elecsys Anti-HAV II is a second generation assay by Roche Diagnostics for the in vitro qualitative detection of total antibodies (IgG and IgM) to the hepatitis A virus (HAV) in human pediatric (ages 2 through 21 years) and adult serum and plasma. It is intended for use on the cobas e 601 immunoassay analyzer. The cobas e family of analyzers employs the electrochemiluminescence "ECLIA" technology. The assay is an 18-minute assay utilizing a competition principle.

Results are determined automatically by the software by comparing the electrochemiluminescence signal obtained from the reaction product of the sample with the signal of the cutoff value previously obtained by calibration.

The reagent rackpack working solutions include:

M: Streptavidin-coated microparticles .
R1: HAV Ag (cell culture) .
R2: Biotinylated monoclonal anti-HAV antibody, monoclonal Anti-HAV antibody . labeled with ruthenium complex
AHAV 2 Cal1: Negative Calibrator 1 (human serum) .
AHAV 2 Cal2: Positive Calibrator 2 (anti-HAV (human), approximately 60 IU/L in . human serum)

PreciControl Anti-HAV II is a ready-for-use control serum based on human serum both in the negative and positive concentration range. The controls are used for monitoring the performance of the Elecsys Anti-HAV II immunoassay. PreciControl Anti-HAV II is sold separately from the Elecsys Anti-HAV II immunoassay reagent.

2. INTENDED USE

Immunoassay for the in vitro qualitative detection of total antibodies (IgG and IgM) to hepatitis A virus (HAV) in human pediatric (ages 2 through 21 years) and adult serum and plasma (Li-Heparin, potassium EDTA, Na-Citrate, Na-Heparin). The assay, in conjunction with other

6

serological and clinical information, is indicated as an aid in the clinical laboratory diagnosis of acute or past hepatitis A virus infection in persons with signs or symptoms of hepatitis and in persons at increased risk for hepatitis A infection, or as an aid to identify HAV susceptible individuals and to determine the presence of an antibody response to HAV in vaccine recipients.

The electrochemiluminescence immunoassay "ECLIA" is intended for use on the cobas e immunoassay analyzers.

Assay performance characteristics have not been established for immunocompromised or immunosuppressed patients. This assay has not been FDA cleared or approved for the screening of blood or plasma donors.

3. TECHNOLOGICAL CHARACTERISTICS

Elecsys Anti-HAV II utilizes electrochemiluminescence "ECLIA" technology for the qualitative detection of total antibodies (IgG and IgM) to the hepatitis A virus (HAV) in human pediatric (ages 2 through 21 years) and adult serum and plasma on the cobas e 601 immunoassay analyzer.

The following tables compare the Elecsys Anti-HAV II with its predicate device, Elecsys Anti-HAV (K100903).

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Assay Comparison Table 1:

Feature	Elecsys Anti-HAV (K100903)	Elecsys Anti-HAV II
Intended Use/Indications for
Use	Immunoassay for the in vitro qualitative
detection of total antibodies (IgM and
IgG) to hepatitis A virus in human serum
and plasma (K2-EDTA). The assay is
intended for use as an aid in the
laboratory diagnosis of past or
acute/recent hepatitis A infection. Assay
results, in conjunction with other
laboratory results and clinical
information, may be used to provide
presumptive evidence of infection with
hepatitis A virus in persons with signs or
symptoms of hepatitis and in persons at
risk for hepatitis A infection, or used as an
aid to determine the presence of antibody
response to HAV in vaccine recipients.
The electrochemiluminescence
immunoassay "ECLIA" is intended for
use on Elecsys and cobas e immunoassay
analyzers.
This assay is not intended for screening
blood or solid or soft tissue donors. Assay
performance characteristics have not been
established for immunocompromised or
immunosuppressed patients. The users are
responsible for establishing their own
assay performance characteristics in these
populations.	Immunoassay for the in vitro qualitative
detection of total antibodies (IgG and
IgM) to hepatitis A virus (HAV) in
human pediatric (ages 2 through 21
years) and adult serum and plasma (Li-
Heparin, potassium EDTA, Na-Citrate,
Na-Heparin). The assay, in conjunction
with other serological and clinical
information, is indicated as an aid in the
clinical laboratory diagnosis of acute or
past hepatitis A virus infection in
persons with signs or symptoms of
hepatitis and in persons at increased risk
for hepatitis A infection, or as an aid to
identify HAV susceptible individuals
and to determine the presence of an
antibody response to HAV in vaccine
recipients.
The electrochemiluminescence
immunoassay "ECLIA" is intended for
use on cobas e immunoassay analyzers.
Assay performance characteristics have
not been established for
immunocompromised or
immunosuppressed patients. This assay
has not been FDA cleared or approved
for the screening of blood or plasma
donors.
Assay Method	Competition principle binding protein	Same
Detection Method	Electrochemiluminescence	Same
Applications/Test Time	18 minutes	Same
Instrument Platform	Elecsys 2010, cobas e 411, cobas e 601,
cobas e 602, and MODULAR
ANALYTICS E170	cobas e 601
Sample volume	50 µL	20 µL
Sample Type/Matrix	Human serum, plasma	Same
Sample Anticoagulants	K2-EDTA	Li-Heparin, potassium EDTA, Na-
Citrate, Na-Heparin
Calibrator	Anti-HAV Cal1 and Cal2 (packed in kit)	Anti-HAV II Cal1 and Cal2 (packed in
kit)
Calibration Method	2-point calibration	Same
Feature	Elecsys Anti-HAV (K100903)	Elecsys Anti-HAV II
Calibration Interval	Calibration must be performed once per
reagent lot using fresh reagent (i.e. not
more than 24 hours since the reagent kit
was registered on the analyzer). Renewed
calibration is recommended as follows:
• after 1 month (28 days) when using
the same reagent lot
• after 7 days (when using the same
reagent kit on the analyzer)
• as required: e.g. quality control
findings outside the defined limits	Same
Controls	PreciControl Anti-HAV	PreciControl Anti-HAV II
Traceability/Standardization	Second International Standard for Anti
Hepatitis A, Immunoglobulin, Human,
NIBSC code: 97/646	Same
Reagent Stability	Store at 2-8°C.
Do not freeze.
Store the Elecsys reagent kit upright in
order to ensure complete availability of
the microparticles during automatic
mixing prior to use.
Unopened at 2-8°C, up to stated
expiration date.
After opening at 2-8°C, 8 weeks.
On the analyzers at 20-25°C, 7 days or 4
weeks when stored alternative in the
refrigerator and on the analyzer, with the
total time onboard on the analyzer not
exceeding 40 hours.	Store at 2-8°C.
Do not freeze.
Store the Elecsys reagent kit upright in
order to ensure complete availability of
the microparticles during automatic
mixing prior to use.
Unopened at 2-8°C, up to stated
expiration date.
After opening at 2-8°C, 8 weeks.
On the analyzers at 20-25°C, 8 weeks.
Feature	Elecsys Anti-HAV (K100903)	Elecsys Anti-HAV II
Limitations	Bilirubin 5 mg/day) until at least 8 hours
following the last biotin administration.
In vitro tests were performed on 18
commonly used pharmaceuticals
(Acetylcystein, Ampicillin, Ascorbic acid,
Ca Dobesilate, Cyclosporine, Cefoxitin,
Heparin, Intralipid, Levodopa,
Methyldopa, Metronidazole,
Phenylbutazone, Tetracycline,
Acetylsalicylic Acid, Rifampicin,
Acetaminophen, Ibuprofen and
Theophylline). No interference with the
assay was found.
In rare cases, interference due to
extremely high titers of antibodies to
analyte specific antibodies, streptavidin or
ruthenium can occur. These effects are
minimized by suitable test design.	Bilirubin 1.0 COI +/- 20% recovery,
≤ 1.0 COI +/- 0.20 recovery
Negative specimens with biotin
concentrations up to 100 ng/mL
demonstrated ≤ 11 % negative bias in
COI values. Biotin concentrations
greater than 100 ng/mL lead to higher
negative bias and in consequence can
lead to false positive Elecsys Anti-HAV
II results. Some studies have shown that
serum concentrations of biotin can reach
up to 355 ng/mL within the first hour
after ingestion for subjects consuming
supplements of 20 mg biotin per day and
up to 1160 ng/mL in plasma for subjects
consuming a single dose of 300 mg
biotin.
In vitro tests were performed on 18
commonly used pharmaceuticals
(Acetylcystein, Ampicillin-Na, Ascorbic
acid, Ca Dobesilate, Cyclosporine,
Cefoxitin, Doxycycline, Heparin,
Levodopa, Methyldopa +1.5,
Metronidazole, Phenylbutazone,
Tetracycline, Acetylsalicylic Acid,
Rifampicin, Acetaminophen, Ibuprofen
and Theophylline). No interference with
the assay was found.
In rare cases, interference due to
extremely high titers of antibodies to
analyte specific antibodies, streptavidin
or ruthenium can occur. These effects are
minimized by suitable test design.

8

9

10

NON-CLINICAL PERFORMANCE EVALUATION 4.

Non-clinical performance evaluation for Elecsys Anti-HAV II is briefly summarized below.

Precision 4.1.

4.1.1. Repeatability and Within-Laboratory Precision

Precision measurements were conducted to evaluate repeatability (within-run precision) and within-laboratory precision (intermediate precision) in a study based on the protocol of CLSI EP05-A3. Precision was evaluated on a single cobas e 601 immunoassay analyzer. One Elecsys Anti-HAV II reagent lot was evaluated. The protocol consisted of testing 2 aliquots each of two levels of control and 5 human sera per run, 2 runs per day for 21 days. Calibration was performed on day 1 and on day 17. Serum samples were human serum sample pools.

| | | Repeatability | | Within-Laboratory
(Intermediate) Precision | | |
|--------------------|------------|---------------|--------|-----------------------------------------------|--------|----|
| Sample | Mean (COI) | SDa)
(COI) | CV (%) | SD
(COI) | CV (%) | n |
| Human serum 1 | 1.42 | 0.016 | 1.1 | 0.028 | 2.0 | 84 |
| Human serum 2 | 1.15 | 0.012 | 1.1 | 0.022 | 1.9 | 84 |
| Human serum 3 | 0.955 | 0.009 | 0.9 | 0.022 | 2.3 | 84 |
| Human serum 4 | 0.665 | 0.009 | 1.3 | 0.020 | 2.9 | 84 |
| Human serum 5 | 0.006 | 0.0002 | 3.0 | 0.0002 | 3.3 | 84 |
| PCb) Anti-HAV II 1 | 1.30 | 0.015 | 1.1 | 0.024 | 1.8 | 84 |
| PC Anti-HAV II 2 | 0.339 | 0.005 | 1.5 | 0.009 | 2.7 | 84 |

Table 2: Repeatability and Within-Laboratory Precision Results

SD = Standard Deviation a)

PC = PreciControl b)

4.2. Analytical Sensitivity

4.2.1. Determination of Cut-off Sensitivity

The cut-off of the Elecsys Anti-HAV II assay was established with internal studies, and the validation of the cut-off was performed by external clinical studies. In order to determine the cut-off sensitivity, the 2nd International Standard for Anti-Hepatitis A, Immunoglobulin, Human,

11

NIBSC code: 97/646 was serially diluted to 10 samples and tested fourfold with three different reagent and calibrator lots. The cut-off of COI = 1.0 corresponds to ≤ 25.4 IU/L.

| Dilution-
factor/
Sample | Target value
[IU/L] | Measuring cell 1 [COI] | | Measuring cell 2 [COI] | | [COI]
Mean |
|--------------------------------|------------------------|------------------------|-------|------------------------|-------|---------------|
| | | Run 1 | Run 2 | Run 1 | Run 2 | |
| Ref-Std_01 | 0 | 1.31 | 1.28 | 1.31 | 1.30 | 1.30 |
| Ref-Std_02 | 10.2 | 1.16 | 1.17 | 1.17 | 1.16 | 1.17 |
| Ref-Std_03 | 20.3 | 1.05 | 1.03 | 1.06 | 1.04 | 1.05 |
| Ref-Std_04 | 30.5 | 0.921 | 0.923 | 0.923 | 0.946 | 0.928 |
| Ref-Std_05 | 59.9 | 0.628 | 0.626 | 0.637 | 0.646 | 0.634 |
| Ref-Std_06 | 99.7 | 0.365 | 0.361 | 0.365 | 0.369 | 0.365 |
| Ref-Std_07 | 200 | 0.098 | 0.098 | 0.104 | 0.102 | 0.100 |
| Ref-Std_08 | 300 | 0.035 | 0.034 | 0.036 | 0.035 | 0.035 |
| Ref-Std_09 | 400 | 0.017 | 0.017 | 0.017 | 0.017 | 0.017 |
| Ref-Std_10 | 500 | 0.011 | 0.011 | 0.019 | 0.011 | 0.013 |
| Cut-off sensitivity | | 24.6 IU/L | | | | |

Table 3: Lot MP01

Table 4: Lot PoQ

| Dilution-
factor/
Sample | Target value
[IU/L] | Measuring cell 1 [COI] | | Measuring cell 2 [COI] | | [COI]
Mean |
|--------------------------------|------------------------|------------------------|-------|------------------------|-------|---------------|
| | | Run 1 | Run 2 | Run 1 | Run 2 | |
| Ref-Std_01 | 0 | 1.28 | 1.28 | 1.27 | 1.28 | 1.28 |
| Ref-Std_02 | 10.2 | 1.14 | 1.15 | 1.17 | 1.17 | 1.16 |
| Ref-Std_03 | 20.3 | 1.05 | 1.06 | 1.04 | 1.04 | 1.05 |
| Ref-Std_04 | 30.5 | 0.941 | 0.944 | 0.934 | 0.942 | 0.940 |
| Ref-Std_05 | 59.9 | 0.663 | 0.663 | 0.674 | 0.665 | 0.667 |
| Ref-Std_06 | 99.7 | 0.389 | 0.399 | 0.399 | 0.401 | 0.397 |
| Ref-Std_07 | 200 | 0.113 | 0.112 | 0.115 | 0.115 | 0.114 |
| Ref-Std_08 | 300 | 0.039 | 0.038 | 0.040 | 0.040 | 0.040 |
| Ref-Std_09 | 400 | 0.018 | 0.018 | 0.018 | 0.019 | 0.018 |
| Ref-Std_10 | 500 | 0.011 | 0.011 | 0.011 | 0.011 | 0.011 |
| Cut-off sensitivity | | | | 25.4 IU/L | | |

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| Dilution-factor/
Sample | Target value
[IU/L] | Measuring cell 1 [COI] | | Measuring cell 2 [COI] | | [COI]
Mean | |
|----------------------------|------------------------|------------------------|-------|------------------------|-------|---------------|--|
| | | Run 1 | Run 2 | Run 1 | Run 2 | | |
| Ref-Std_01 | 0 | 1.25 | 1.25 | 1.26 | 1.27 | 1.26 | |
| Ref-Std_02 | 10.2 | 1.11 | 1.10 | 1.13 | 1.15 | 1.12 | |
| Ref-Std_03 | 20.3 | 0.976 | 0.975 | 0.982 | 0.984 | 0.980 | |
| Ref-Std_04 | 30.5 | 0.867 | 0.859 | 0.873 | 0.872 | 0.868 | |
| Ref-Std_05 | 59.9 | 0.551 | 0.548 | 0.563 | 0.559 | 0.555 | |
| Ref-Std_06 | 99.7 | 0.269 | 0.274 | 0.276 | 0.276 | 0.274 | |
| Ref-Std_07 | 200 | 0.048 | 0.048 | 0.049 | 0.048 | 0.048 | |
| Ref-Std_08 | 300 | 0.016 | 0.016 | 0.016 | 0.016 | 0.016 | |
| Ref-Std_09 | 400 | 0.010 | 0.010 | 0.010 | 0.010 | 0.010 | |
| Ref-Std_10 | 500 | 0.009 | 0.009 | 0.009 | 0.009 | 0.009 | |
| Cut-off sensitivity | | 20.1 IU/L | | | | | |

Table 5: Lot P02

4.3. Human Anti-Mouse Antibodies (HAMA)

The effect on detection of analyte in the presence of HAMA using the Elecsys Anti-HAV II was determined on the cobas e 601 immunoassay analyzer using 11 human serum samples. Only samples that are double positive for HAMA and anti-HAV antibodies were used, since HAMA interference would lead to false negative results in the competitive assay format. No HAMA interference was found within the predefined acceptance criteria.

Endogenous Interferences 4.4.

Hemoglobin/Bilirubin/Lipemia 4.4.1.

The purpose of this study was to evaluate endogenous substances for potential interference with the parameters measured on the Elecsys Anti-HAV II on the cobas e 601 immunoassay analyzer. For each interfering substance, four human serum samples were tested in accordance with CLSI EP07-A2.

4.4.2. Rheumatoid Factor Interference

The recovery of analyte values in the presence of endogenous interfering substances was determined with the Elecsys Anti-HAV II on the cobas e 601 immunoassay analyzer.

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One aliquot of each serum sample was spiked with the interfering substance, and another aliquot with the same volume of the solvent (buffer matrix) of the interfering substance (dilution pool). The interfering pool was then diluted into the dilution pool in 10% increments.

The recovery for each sample was calculated by comparison to the reference sample.

Serum Albumin, IgG, IgA, and IgM Interference 4.4.3.

One aliquot of each serum sample was spiked with the interfering substance (lyophilisate). In this case, another aliquot without any additives (since the interfering substance is a lyophilisate) was used as dilution pool. The interfering pool was then diluted into the dilution pool in 14.3% increments.

The deviation or recovery for each sample was calculated by comparison to the reference sample.

Conclusion:

All results met the pre-defined acceptance criteria, demonstrating no interference from endogenous substances up to the levels shown in the table below.

| Interferent | Interfering substance
measured up to | No interference
seen up to |
|-----------------------|-----------------------------------------|-------------------------------|
| Intralipid® (Lipemia) | 2000 mg/dL | 2000 mg/dL |
| Bilirubin | 66 mg/dL | 66 mg/dL |
| Hemoglobin | 1000 mg/dL | 1000 mg/dL |
| Rheumatoid Factor | 2000 IU/mL | 1400 IU/mL |
| human Serum albumin | 7.00 g/dL | 7.00 g/dL |
| human IgG | 7.00 g/dL | 7.00 g/dL |
| human IgM | 1.00 g/dL | 1.00 g/dL |
| human IgA | 1.60 g/dL | 1.60 g/dL |

Table 6: Summary of Results - Endogenous Interfering Substances

4.5. Biotin

The purpose of this study was to evaluate biotin for potential interference with the parameters measured on the Elecsys Anti-HAV II on the cobas e 601 immunoassay analyzer. Five human serum samples were tested in accordance with CLSI EP07-A2. Negative specimens with biotin

14

concentrations up to 100 ng/mL demonstrated ≤11% negative bias in COI values. Biotin concentrations greater than 100 ng/mL lead to higher negative bias and in consequence can lead to false positive Elecsys Anti-HAV II results. Refer to Limitations for additional information on biotin interference.

4.6. Analytical Specificity/Cross-Reactivity

The effect on detection of analyte in the presence of potential cross-reacting antibodies using the Elecsys Anti-HAV II was determined on the cobas e 601 immunoassay analyzer with native human serum and plasma sample pools.

Anti-HAV negative samples containing potential cross-reacting antibodies to other infectious diseases were measured with Elecsys Anti-HAV and Elecsys Anti-HAV II. In total, 10 samples with acute Hepatitis B infection, 10 samples with acute Hepatitis C infection, 10 samples with HIV infection, 10 samples with EBV infection, 10 samples with Anti-CMV antibodies, 10 samples with Anti-HSV antibodies, 10 samples with Toxoplasma Gondii infection, 10 samples with Treponema Pallidum infection, 10 samples with Anti-Mumps/Rubeola antibodies, 10 samples with Anti-Rubella antibodies, 10 samples with Anti-Parvovirus B19 antibodies and 10 samples with ANA Autoimmune antibodies were tested.

No cross-reactivity with other infectious agents was found.

Exogenous Interferences – Drugs 4.7.

The recovery of analyte values in the presence of drugs was determined by comparing values obtained from samples spiked with 18 commonly used pharmaceutical compounds (Acetylcystein, Ampicillin-Na, Ascorbic acid, Ca-Dobesilate, Cyclosporine, Cefoxitin, Doxycycline, Heparin, Levodopa, Methyldopa +1.5, Metronidazole, Phenylbutazone, Tetracycline, Acetylsalicylic Acid, Rifampicin, Acetaminophen, Ibuprofen and Theophylline) with the reference sample (unspiked). Four human serum samples (native human serum pools) were used and tested on the cobas e 601 immunoassay analyzer according to CLSI EP07-A2. The drug concentrations tested correspond at least to the five times maximum daily doses. The four serum samples were divided into aliquots and spiked with the potential interferents. The reference sample without interferent was spiked with the respective amount of solvent only.

15

The Elecsys Anti-HAV II concentration (as COI) of the spiked aliquots was determined in threefold determination and compared to the Elecsys Anti-HAV II result determined for the reference aliquot (also in three-fold determination) on one cobas e 601 immunoassay analyzer.

All results met the pre-defined acceptance criteria, demonstrating no interference from the drug substances at the tested concentrations.

Sample Matrix Comparison 4.8.

The recovery of analyte values in the presence of anticoagulants with the Elecsys Anti-HAV II was determined on the cobas e 601 immunoassay analyzer by comparing Elecsys Anti-HAV II results obtained from samples drawn into serum and plasma collection tubes. The samples were spiked with anti-HAV positive samples from individual donors to obtain a range of anti-HAV concentrations. The recovery of each plasma sample to the matching serum sample was calculated. At least 60 serum/plasma pairs were tested for each kind of anticoagulant in single determination (K2-EDTA, K3-EDTA, Na-Heparin, Li-Heparin, Na-Citrate).

The specifications were met for all anti-coagulants, demonstrating that K2-EDTA, K3-EDTA, Na-Heparin, Li-Heparin, and Na-Citrate are acceptable sample types for use with Elecsys Anti-HAV II.

Analytical Method Comparison to Predicate 4.9.

A method comparison of Elecsys Anti-HAV II on the cobas e 601 immunoassay analyzer versus the predicate device, Elecsys Anti-HAV, was conducted according to CLSI EP09-A3.

A total of 215 modified serum samples (≥100 negative and ≥100 positive samples) were measured in singleton on cobas e 601 immunoassay analyzer. The samples were primarily prepared by mixing negative and positive sera from individual donors.

Results for the method comparison between Elecsys Anti-HAV II and Elecsys Anti-HAV met the pre-defined acceptance criteria, which demonstrates equivalency between the candidate and predicate devices.

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Table 7: Analytical Method Comparison Agreement Rates Between Elecsys Anti-HAV II and Elecsys Anti-HAV

Elecsys Anti-HAV II Result	Elecsys Anti-HAV Result
	Reactive	Borderline	Non-Reactive
Reactive	107	2	0
Non-Reactive	0	8	98
Total	107	10	98

The positive percent agreement rate was 100% (107/107), the negative percent agreement rate was 100% (98/98). Of ten samples with results in the borderline zone of the predicate device, Elecsys Anti-HAV, 20% (2/10) were positive and 80% (8/10) were negative by the Elecsys Anti-HAV II.

4.10. Reagent Stability

The reagent stability testing was performed in two different studies:

Study 1: Reagent stability after first opening at 2-8°C (8 weeks) .
Study 2: On-board reagent stability (8 weeks) .

4.10.1. Reagent Stability After First Opening

Elecsys Anti-HAV II reagent kits can be used after first opening for up to 8 weeks when stored at 2-8°C. Reagent stability after first opening for the Elecsys Anti-HAV II assay was tested on one cobas e 601 immunoassay analyzer.

A fresh reagent rackpack was placed on the analyzer and calibrated. Reference values for the samples tested were determined. After initial measurement the kit was removed from the analyzer and kept at 2-8 °C for 35 and 63 days. After 35 and 63 days, the kit was placed on the analyzer again, calibrated, and the test samples were determined. Recovery was calculated based on the result at time-point Day 0. Two control samples and seven samples were tested in duplicate.

All pre-defined acceptance criteria were met, demonstrating reagent stability for 8 weeks after first opening at 2-8°C.

17

4.10.2. On-Board Reagent Stability

Elecsys Anti-HAV II reagent kits can be stored on-board the analyzers for up to 8 weeks. A new calibration of the kit kept on-board is recommended every 7 days.

Reagent on-board stability for the Elecsys Anti-HAV II assay was tested on one cobas e 601 immunoassay analyzer. A fresh reagent rackpack was placed on the analyzer and calibrated. All samples were measured on day 0. On day 22, 29, 35, 49 and 63, the same samples were measured with the same reagent kit kept at 20°C ± 3°C (on-board condition) using the calibration curves established on day 0, 22, 29, 35, 49 and 63, respectively. Recovery was calculated based on the result at time-point Day 0. Two control samples were tested in singleton and seven samples were tested in duplicate.

All pre-defined acceptance criteria were met, demonstrating reagent on-board stability for 8 weeks on the cobas e 601 immunoassay analyzer.

4.11. Calibration Stability

To test calibration stability, two studies were completed, including:

Study 1: Lot calibration stability .
Study 2: On-board calibration stability .

4.11.1. Lot Calibration Stability

Calibration of an Elecsys Anti-HAV II reagent lot is recommended every 28 days (4 weeks). During that time period, fresh reagent kits of the same lot can be used without calibration using the calibration curve of the Day 0 reagent kit.

Elecsys Anti-HAV II was calibrated with a fresh reagent kit on day 0 using a cobas e 601 immunoassay analyzer. After 35 days (5 weeks), a new reagent kit of the same lot was used and recovery of samples was determined using the calibration curve of Day 0. Two control samples were tested in singleton and seven human serum samples were tested in duplicate.

All pre-defined acceptance criteria were met, demonstrating renewed calibration is recommended after 4 weeks when using the same reagent lot.

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4.11.2. On-Board Calibration Stability

On-board calibration stability for the Elecsys Anti-HAV II assay was tested on one cobas e 601 immunoassay analyzer. A fresh reagent rackpack was placed on the analyzer and calibrated. All samples were measured on day 0. On day 8, the same samples were measured with the same reagent kit kept at 20°C ± 3°C (on-board condition) using the calibration curves established on day 1. Two control samples were tested in singleton and seven human serum samples were tested in duplicate.

All pre-defined acceptance criteria were met, demonstrating that a calibration can be used for up to one week when using the same lot of reagents stored on the analyzer.

4.12. Sample Stability

To test sample stability, four studies were completed, including:

Study 1: Sample stability at 2-8°C .
Study 2: Sample stability at room temperature (15-25°C) .
Study 3: Sample stability at -15 to -25°C .
Study 4: Freeze/thaw cycles .

4.12.1. Sample Stability at 2-8°C

Six human serum, K2-EDTA, Li-Heparin, and Na-Citrate plasma samples and seven K3-EDTA and Na-Heparin plasma samples were aliquoted and measured fresh (reference value) and after storage at 2-8°C for 14 days. Measurements were performed with three-fold determination on a cobas e 601 immunoassay analyzer and recovery was calculated as percent of the reference value.

All pre-defined acceptance criteria were met, demonstrating the specimens are stable for 14 days at 2-8°C.

4.12.2. Sample Stability at Room Temperature (15-25°C)

Six human serum, K2-EDTA, Li-Heparin, and Na-Citrate plasma samples and seven K3-EDTA and Na-Heparin plasma samples were aliquoted and measured fresh (reference value) and after

19

storage at 15-25°C for 6 days. Measurements were performed with three-fold determination on a cobas e 601 immunoassay analyzer and recovery was calculated as percent of the reference value.

All pre-defined acceptance criteria were met, demonstrating the specimens are stable for 6 days at 15-25°C.

4.12.3. Sample Stability at -15 to -25°C

Six human serum, K2-EDTA, Li-Heparin, and Na-Citrate plasma samples and seven K3-EDTA and Na-Heparin plasma samples were aliquoted and measured fresh (reference value) and after storage at -15 to -25℃ for 3 months. Measurements were performed with three-fold determination on a cobas e 601 immunoassay analyzer and recovery was calculated as percent of the reference value.

All pre-defined acceptance criteria were met, demonstrating the specimens are stable for 3 months at -15 to -25°C.

4.12.4. Sample Stability – Freeze/Thaw Cycles

Six human serum, K2-EDTA, Li-Heparin, and Na-Citrate plasma samples and seven K3-EDTA and Na-Heparin plasma samples were aliquoted and measured fresh (reference value) and after five freeze/ thaw cycles. Measurements were performed with three-fold determination on a cobas e 601 immunoassay analyzer and recovery was calculated as percent of the reference value.

All pre-defined acceptance criteria were met, demonstrating the specimens are stable for 5 freeze/thaw cycles.

EXTERNAL (CLINICAL) TESTING 5.

5.1. Reproducibility

Precision results were collected on three different cobas e 601 immunoassay analyzers across three external testing sites utilizing a single lot of Elecsys Anti-HAV II reagent. Four human serum pools and two Elecsys Anti-HAV II PreciControl materials were tested in three replicates

20

per run, two runs per day for a 5-day format according to CLSI EP05-A3. All testing was performed on the same analyzer with a new rackpack for each day. Repeatability, withinlaboratory, and reproducibility were calculated as SD and CV values.

Ten dummy specimens were tested in each run, as well as between runs, so as to separate the runs within each day. The data were evaluated by Component of Variance Analysis.

| | | | | | Repeatability | | Between
Run | | Between
Day | | Between
Site | | Reproducibility | | |
|----------------------|----|-------|--------|-------|---------------|-------------------|------------------------|-------|----------------|-------|-----------------|-------|-----------------|-------------------|------------------------|
| Specimen
Material | N | Mean | Median | Min | Max | SD
(95%
CI) | %
CV
(95%
CI) | SD | %
CV | SD | %
CV | SD | %
CV | SD
(95%
CI) | %
CV
(95%
CI) |
| HSP 01 | 90 | 0.813 | 0.814 | 0.764 | 0.856 | 0.010
(0.012) | 1.3
(1.5) | 0.016 | 1.9 | 0.000 | 0.0 | 0.011 | 1.3 | 0.022
(0.031) | 2.7
(3.8) |
| HSP 02 | 90 | 0.904 | 0.908 | 0.846 | 0.953 | 0.012
(0.014) | 1.3
(1.5) | 0.016 | 1.8 | 0.000 | 0.0 | 0.014 | 1.5 | 0.024
(0.036) | 2.7
(4.0) |
| HSP 03 | 90 | 1.009 | 1.010 | 0.934 | 1.070 | 0.018
(0.021) | 1.7
(2.1) | 0.013 | 1.3 | 0.004 | 0.4 | 0.016 | 1.6 | 0.027
(0.041) | 2.7
(4.1) |
| HSP 04 | 90 | 0.449 | 0.450 | 0.408 | 0.478 | 0.005
(0.006) | 1.1
(1.3) | 0.011 | 2.5 | 0.000 | 0.0 | 0.007 | 1.5 | 0.014
(0.020) | 3.1
(4.4) |
| PC A-
HAV2_L1 | 90 | 1.299 | 1.300 | 1.230 | 1.350 | 0.018
(0.022) | 1.4
(1.7) | 0.009 | 0.7 | 0.010 | 0.7 | 0.018 | 1.4 | 0.029
(0.046) | 2.2
(3.5) |
| PC A-
HAV2_L2 | 90 | 0.376 | 0.375 | 0.347 | 0.410 | 0.006
(0.007) | 1.6
(1.9) | 0.013 | 3.3 | 0.000 | 0.0 | 0.003 | 0.9 | 0.014
(0.018) | 3.8
(4.8) |

Table 8: Results - Elecsys Anti-HAV II and PreciControls Anti-HAV II on cobas e 601 Immunoassay Analyzer (all sites combined)

One-sided upper 95% confidence interval displayed

5.2. Method Comparison Versus Predicate

The Elecsys Anti-HAV II was performed at each of the three clinical laboratories with a single lot of reagent to assess the performance of the assay in a testing environment that most closely resembles that of the final user. The objective of this study was to provide data for US- and non-US-obtained specimens by testing the Elecsys Anti-HAV II on the cobas e 601 immunoassay analyzer and comparing the results to an FDA-cleared predicate assay. The Elecsys Anti-HAV assay was used as the predicate device for comparison at each of the three laboratories. The study included specimens from patients in the following cohorts: routine HAV testing, hospitalized, increased risk, symptomatic, characterized acute HAV infected, and pediatric.

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	Elecsys Anti-HAV Assay Results (all sites combined)
Elecsys Anti-HAV
II Results	Reactive
$\geq$ 22 IU/L	Border 18.0 $\leq$ IU/L 1.0	0	1	437
Total	501	7	453	961

Table 9: Method Comparison Results (all sites combined)

	Absolute	Relative %	95 % CI
PPAc)	501/502	99.80	98.90; 99.99
NPAd)	437/459	95.21	92.83; 96.97

c) PPA = Positive Percent Agreement

NPA = Negative Percent Agreement d)

Positive percent agreement and negative percent agreement with their respective 95th percentile confidence interval (CI) for each cohort are summarized in the following table. The lower end of the 95% confidence intervals for PPA ranged from 90.45% in the hospitalized cohort to 97.18% in the symptomatic cohort. The lower end of the ≥ 95% confidence intervals for NPA ranged from 52.36% in the pediatric cohort to 93.04% in the hospitalized cohort. For the pediatric cohort, there were 14 specimens negative by both the predicate Elecsys Anti-HAV assay and the Elecsys Anti-HAV II assay. There were 4 discordant results, of which 2 were borderline with the predicate assay but positive with the Elecsys Anti-HAV II assay and 2 were negative with the predicate assay but positive with the Elecsys Anti-HAV II assay. Discordant results were counted against Elecsys Anti-HAV II when calculating agreement (77.8%). The lower bound of the 95% CI for the NPA in the pediatric population is influenced by the small number of specimens (n = 18). The overall percentages for all cohorts combined met the expected performance of a lower bound of the 95% confidence interval of ≥ 90% for the agreement rates (positive and negative) versus the predicate assay (Elecsys Anti-HAV assay). The lower limit of the confidence interval for the PPA was 98.90%, and the lower limit of the confidence interval for NPA was 92.83%.

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| Summary of the percent agreements for various specimen cohorts: Elecsys Anti-HAV IIe) assay

versus the predicate (Elecsys Anti-HAV assay)f)
Cohort	PPA		NPA
	PPA (x/n)	95 % CI	NPA (x/n)	95 % CI
Routine HAV testing	100 (91/91)	(96.03, 100)	94.50 (103/109)	(88.40, 97.95)
Hospitalized	98.21 (55/56)	(90.45, 99.95)	97.22 (140/144)	(93.04, 99.24)
Increased risk for hepatitis	100 (119/119)	(96.95, 100)	94.25 (82/87)	(87.10, 98.11)
Symptomatic	100 (129/129)	(97.18, 100)	96.70 (88/91)	(90.67, 99.31)
Characterized acute HAV	100 (65/65)	(94.48, 100)	100 (10/10)	(69.15, 100)
Pediatric	100 (42/42)	(91.59, 100)	77.78 (14/18)	(52.36, 93.59)
Overall	99.80 (501/502)	(98.90, 99.99)	95.21 (437/459)	(92.83, 96.97)

Table 10: Percent Agreements for the Various Specimen Cohorts

e) Cutoff of 1.0 COI used for Elecsys Anti-HAV II assay

f) Specimens with results in the borderline range of (18.0 ≤ IU/L