Zeus IFA ANA HEp-2 Test System, Zeus dIFine

K201956 · Zeus Scientific, Inc. · DHN · Apr 29, 2022 · Immunology

Device Facts

• Record ID: K201956
• Device Name: Zeus IFA ANA HEp-2 Test System, Zeus dIFine
• Applicant: Zeus Scientific, Inc.
• Product Code: DHN · Immunology
• Decision Date: Apr 29, 2022
• Decision: SESE
• Submission Type: Traditional
• Regulation: 21 CFR 866.5100
• Device Class: Class 2

Indications for Use

ZEUS IFA™ ANA HEp-2 Test System is an indirect immunofluorescence assay for the qualitative detection and semiquantitative determination of IgG anti-nuclear antibodies in human serum by manual fluorescence microscopy or with ZEUS dlFine®. The presence of anti-nuclear antibodies can be used in conjunction with other serological tests and clinical findings to aid in the diagnosis of systemic lupus erythematosus and other systemic rheumatic diseases. All suggested results obtained with ZEUS dIFine® must be confirmed by a trained operator. ZEUS dIFine® is an automated instrument consisting of a fluorescent microscope and software that acquires, interprets, stores and displays digital images of stained indirect immunofluorescence slides. ZEUS dlFine can only be used with FDA cleared or approved ZEUS in vitro diagnostic assays that are indicated for use on this instrument. All suggested results obtained with ZEUS dIFine must be confirmed by a trained operator

Device Story

ZEUS IFA ANA HEp-2 Test System uses indirect immunofluorescence (IFA) to detect IgG anti-nuclear antibodies in human serum. ZEUS dIFine is an automated digital fluorescence microscope and software system that acquires, interprets, stores, and displays digital images of stained IFA slides. The system is operated by trained laboratory personnel in a clinical setting. The software processes images to provide suggested results, which must be confirmed by a trained operator. The device aids clinicians in diagnosing systemic lupus erythematosus and other systemic rheumatic diseases by providing standardized digital interpretation of fluorescence patterns, potentially improving consistency and efficiency compared to manual microscopy.

Clinical Evidence

Clinical performance evaluated using 190 samples from patients with ANA-associated autoimmune diseases (SARDs) and 160-190 samples from non-ANA-associated disease cohorts across three sites. Performance compared manual microscopy (Method A) against automated imaging (Method B) and automated software interpretation (Method C). Results showed high qualitative agreement (PPA 93.7-99.0%, NPA 87.1-96.9%) and endpoint titer agreement (99.2-99.3% within one dilution) across methods. Clinical sensitivity and specificity were consistent across all three interpretation methods.

Technological Characteristics

System includes HEp-2 substrate slides, FITC-labeled goat anti-human IgG conjugate, and controls. ZEUS dIFine hardware: Olympus fluorescence microscope, 4X/20X objectives, motorized stage (8-slide capacity), LED illumination, and digital camera. Connectivity: Industrial PC with Windows 10. Software: dIFine v1.5.28. Sterilization: Not applicable (reagents). Sensing: FITC light intensity measurement.

Indications for Use

Indicated for qualitative detection and semiquantitative determination of IgG anti-nuclear antibodies (ANA) in human serum to aid in diagnosis of systemic lupus erythematosus and other systemic rheumatic diseases. For prescription use by trained laboratory personnel.

Regulatory Classification

Identification

An antinuclear antibody immunological test system is a device that consists of the reagents used to measure by immunochemical techniques the autoimmune antibodies in serum, other body fluids, and tissues that react with cellular nuclear constituents (molecules present in the nucleus of a cell, such as ribonucleic acid, deoxyribonucleic acid, or nuclear proteins). The measurements aid in the diagnosis of systemic lupus erythematosus (a multisystem autoimmune disease in which antibodies attack the victim's own tissues), hepatitis (a liver disease), rheumatoid arthritis, Sjögren's syndrome (arthritis with inflammation of the eye, eyelid, and salivary glands), and systemic sclerosis (chronic hardening and shrinking of many body tissues).

Predicate Devices

• ZEUS IFA ANA HEp-2 Test System (K152675)
• ZEUS dIFine (K191536)

Submission Summary (Full Text)

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FDA

U.S. FOOD & DRUG

ADMINISTRATION

# 510(k) SUBSTANTIAL EQUIVALENCE DETERMINATION DECISION SUMMARY

ASSAY AND INSTRUMENT

## I Background Information:

A 510(k) Number

K201956

B Applicant

Zeus Scientific, Inc.

C Proprietary and Established Names

Zeus IFA ANA HEp-2 Test System, Zeus dIFine

D Regulatory Information

| Product Code(s) | Classification | Regulation Section | Panel |
| --- | --- | --- | --- |
| DHN | Class II | 21 CFR 866.5100 - Antinuclear Antibody Immunological Test System | IM - Immunology |
| PIV | Class II | 21 CFR 866.4750 - Automated indirect immunofluorescence microscope and software-assisted system | IM - Immunology |

## II Submission/Device Overview:

A Purpose for Submission:

Assay and Instrument

B Measurand:

Anti-nuclear antibodies (ANA)

C Type of Test:

Qualitative and semi-quantitative, indirect immunofluorescence assay (IFA)

Food and Drug Administration

10903 New Hampshire Avenue

Silver Spring, MD 20993-0002

www.fda.gov

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K201956 - Page 2 of 26

## III Intended Use/Indications for Use:

### A Intended Use(s):

See Indications for Use below.

### B Indication(s) for Use:

**Assay:**

ZEUS IFA ANA HEp-2 Test System is an indirect immunofluorescence assay for the qualitative detection and semi-quantitative determination of IgG anti-nuclear antibodies in human serum by manual fluorescence microscopy or with ZEUS dIFine. The presence of anti-nuclear antibodies can be used in conjunction with other serological tests and clinical findings to aid in the diagnosis of systemic lupus erythematosus and other systemic rheumatic diseases. All suggested results obtained with ZEUS dIFine must be confirmed by a trained operator.

**Instrument:**

ZEUS dIFine is an automated instrument consisting of a fluorescent microscope and software that acquires, interprets, stores and displays digital images of stained indirect immunofluorescence slides. ZEUS dIFine can only be used with FDA cleared or approved ZEUS in vitro diagnostic assays that are indicated for use on this instrument. All suggested results obtained with ZEUS dIFine must be confirmed by a trained operator.

### C Special Conditions for Use Statement(s):

Rx - For Prescription Use Only

This device is only for use with reagents that are indicated for use with the device.

The device is for use by a trained operator in a clinical laboratory setting.

All software-aided results must be confirmed by the trained operator.

### D Special Instrument Requirements:

For use only with Zeus dIFine

## IV Device/System Characteristics:

### A Device Description:

ZEUS IFA ANA HEp-2 Test System is an indirect immunofluorescence assay for the qualitative detection and semi-quantitative determination of IgG anti-nuclear antibodies (ANA) in human serum by manual fluorescence microscopy or with ZEUS dIFine.

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K201956 - Page 3 of 26

# ZEUS IFA ANA HEp-2 Test System Assay kit components

1. ANA Hep-2 Substrate slides: Twenty, 12-well slides with absorbent blotter and desiccant pouch.
2. Conjugate: Goat anti-human IgG labeled with fluorescein isothiocyanate (FITC). Contains phosphate buffer with BSA and counterstain. One bottle, 12 mL, white-capped. Ready to use.
3. Positive Control (Human Serum): Will produce positive apple-green, homogeneous, staining of the cell nucleus. One vial, 0.5 mL, red-capped. Ready to use.
4. Negative Control (Human Serum): Will produce no detectable nuclear staining. One vial, 0.5 mL, green-capped. Ready to use.
5. Zorba-NS Sample Diluent: Phosphate-buffered-saline. Four bottles, 30 mL, green-capped. Ready to use.
6. Phosphate-buffered-saline (PBS): pH 7.2 ± 0.2. Ten packets, each sufficient to prepare one liter.
7. Mounting Media (Buffered Glycerol): One clear bottle, 12 mL, clear-capped. Ready to use.

# ZEUS dIFine

The ZEUS dIFine System is an automated fluorescence microscope. It does not process samples or perform the assay, but acquires digital images of representative areas of the ZEUS ANA HEp-2 Test System Assay (IFA) slides. The instrument hardware includes the microscope, camera, microscope control unit, and an LED illumination unit. The ZEUS dIFine System includes an Olympus fluorescence microscope with 4X and 20X objectives. The microscope has a motorized slide stage that accommodates up to eight slides. ZEUS dIFine System includes an industrial computer with Windows 10 operating system as well as dIFine software v 1.5.28.

## B Principle of Operation:

ZEUS IFA ANA HEp-2 Test System is an indirect immunofluorescence assay for the qualitative detection and semi-quantitative determination of IgG anti-nuclear antibodies in human serum by manual fluorescence microscopy or by ZEUS dIFine. The ZEUS IFA ANA HEp-2 Test System detects the presence of circulating ANA in human sera that bind to HEp-2 cells coated on the glass slide. Goat anti-human immunoglobulin-FITC conjugate visualizes the ANA bound to the cells. Any positive reactions will appear as apple-green colored fluorescent staining within the cell. If the sample does not contain ANA, there will be no distinct staining of the cell.

**Manual Interpretation:** The interpretation of the results depends on the pattern observed as well as the titer of the autoantibody present in the specimen. A positive reaction is the presence of any pattern of nuclear apple-green staining observed at a 1:40 dilution based on a 1+ to 4+ scale of staining intensity where 1+ is considered a weak reaction and 4+ a strong reaction. The sponsor recommends sera positive at 1:40 should be titered to endpoint dilution by making 1:40, 1:80, 1:160, etc. serial dilutions. The endpoint titer is the highest dilution that produces a 1+ positive reaction.

**Zeus dIFine Interpretation:** When slides are analyzed by dIFine, digital images of representative fields of view of the well are captured. The default scanning area is composed of 12 fields with each field approximately 610 µm × 510 µm using the 20X objective. All images are taken through a fluorescein isothiocyanate (FITC) filter. The ZEUS dIFine System reads the slides and measures the FITC light intensity of the cells that are included in the region. Then, the ZEUS dIFine System reports the measured average nuclear fluorescence intensity as an index percentage and recommends a qualitative result, and a pattern if it is one of the eight patterns

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recognized by the software which will be confirmed by a human reader. To facilitate the interpretation, the ZEUS dIFine System presents acquired digital images of the slide and the following information for human analysis:

- Negative, positive, or uncertain (only for Zeus IFA ANA HEp-2 Test System on ZEUS dIFine with automated reading (software interpretation) of the slides) classification
- Positive Pattern Information: Zeus dIFine can suggest eight patterns - homogeneous, nucleolar, centromere, speckled, nuclear membrane, nuclear dots, cytoplasmic (ribosomal), cytoplasmic (mitochondrial).
- Image Atlas containing a gallery of mitotic figures (on positive samples of identified patterns).

Trained operators then review the images taken by ZEUS dIFine System. During the review process, further options include:

- Navigation of digitized well using virtual microscope tools (zooming/browsing images at different magnifications).
- Enlargement of images to examine detail.
- Image Atlas to assist with identification of patterns.

The trained operator can confirm the results by clicking the "Validate" button on the screen and accepting the classification (negative/positive and pattern) suggested by the ZEUS dIFine System, or they can revise the suggested ZEUS dIFine classification (negative to positive and vice versa), add comments (if any) and eventually click the "Validate" button on the screen.

## C Instrument Description Information:

1. Instrument Name:
Zeus dIFine

2. Specimen Identification:
Manual Entry or Integrated Barcode slide scanner

3. Specimen Sampling and Handling:
Not applicable

4. Calibration:
Not applicable

5. Quality Control:
Not applicable.

K201956 - Page 4 of 26

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V Substantial Equivalence Information:

A Predicate Device Name(s):

Antinuclear Antibody Test System Tissue Cell Culture Slide

B Predicate 510(k) Number(s):

K781516

C Comparison with Predicate(s):

| Device & Predicate Device(s): | K201956 | K781516 |
| --- | --- | --- |
| Device Trade Name | Zeus IFA ANA HEp-2 Test System, Zeus dIFine | Zeus IFA ANA HEp-2 Test System - ANA Test System Tissue Cell Culture Slide |
| General Device Characteristic Similarities | | |
| Intended Use/Indications For Use | ZEUS IFA ANA HEp-2 Test System is an indirect immunofluorescence assay for the qualitative detection and semi-quantitative determination of IgG anti-nuclear antibodies in human serum by manual fluorescence microscopy or with ZEUS dIFine. The presence of anti-nuclear antibodies can be used in conjunction with other serological tests and clinical findings to aid in the diagnosis of systemic lupus erythematosus and other systemic rheumatic diseases. All suggested results obtained with ZEUS dIFine must be confirmed by a trained operator. | The ZEUS IFA ANA HEp-2 Test System is a pre-standardized assay designed for the qualitative and semi-quantitative detection of antinuclear antibodies. The test is intended to aid in determining SLE and differentiating clinically similar connective tissue disorders and is for In Vitro diagnostic use. |
| Methodology | Indirect immunofluorescence assay | Same |
| Results | Pattern and titer Qualitative and semi-quantitative | Same |
| Sample Matrix | Serum | Same |
| Fluorescence Marker | FITC | Same |
| Control | One positive control and one negative control | Same |
| Screening Dilution | 1:40 | Same |
| Antigen | Hep-2 cells | Same |

K201956 - Page 5 of 26

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SIides | 12-well coated with antigen | Same
Storage Conditions | Unopened test system, mounting media, Conjugate Zorba-NS, Slides, Positive control and Negative control must be stored at 2-8°C | Same
Counterstain | Evans Blue | Same
General Device Characteristic Differences
Interpretation of results | Manual fluorescence microscopy or Zeus dIFine automated microscopy with trained operator verification | Manual fluorescence microscopy

VI Standards/Guidance Documents Referenced:

- CLSI EP05-A3 Evaluation of Precision Performance of Quantitative Measurement Methods, Approved Guideline - 3rd Edition
- CLSI EP06, 2nd ed. Evaluation of the Linearity of Quantitative Measurement Procedures – Second Edition
- CLSI EP07-A2 Interference Testing in Clinical Chemistry, 2nd Edition
- CLSI EP17-A2 Evaluation of Detection Capability for Clinical Laboratory Measurement Procedures, 2nd Edition
- CLSI EP28-A3c Defining, Establishing and Verifying Reference Intervals in the Clinical Laboratory
- IEC 61010-1 Safety Requirements for Electrical Equipment for Measurement, Control, and Laboratory Use Part 1: General Requirements
- ISO 14971 - Medical Devices - Application of risk management to medical devices, Second Edition
- Guidance for Industry and FDA Staff - Guidance for the Content of Premarket submission for Software Contained in Medical Devices (May 11, 2005)

VII Performance Characteristics (if/when applicable):

All analytical and clinical studies were evaluated by comparing the three possible reading methods (A, B, and C) described in the table below; these methods are consistent throughout this document. Method A (i.e., manual imaging and manual reading of the slides with a traditional fluorescence microscope) is considered the reference method (predicate) to which all results are compared. All results generated by the Zeus IFA ANA HEp-2 Test System must be confirmed by a trained operator.

| Interpretation Methods | | | |
| --- | --- | --- | --- |
| Method | Processing | Imaging | Reading/Evaluation of Slides |
| A (predicate) | Manual | Manual | Manual (read of microscope field) |
| B | Automated | Automated | Manual (read of digital image) |
| C | Automated | Automated | Automated (software interpretation) |

K201956 - Page 6 of 26

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The Zeus dIFine identifies the following immunofluorescence (IF) patterns: homogenous, speckled, centromere, nucleolar, nuclear dots, nuclear membrane, cytoplasmic (Ribosomal) and cytoplasmic (Mitochondrial).

## A Analytical Performance:

### 1. Precision/Reproducibility:

#### a. Repeatability - 10-Day Within-Laboratory Precision Study

A low positive serum sample (~1:40 endpoint), a mid-positive serum sample (~1:160 to 1:320 endpoint) and a strong positive serum sample (>1:640 endpoint) were identified for each of the eight patterns reported by dIFine. One ANA negative specimen was also included bringing the group of specimens to a total of 25. These 25 specimens were diluted at 1:40 and assayed in triplicate, on ten different days producing 30 results per sample at one site and by one reader. The slides were interpreted by all three methods for qualitative results. The pattern was determined by all methods for all positive samples.

The study produced 30 results for each of the 25 specimens for each of the three interpretation methods (Method A, Method B and Method C). The results of the qualitative and pattern agreement for within-method and between-methods are evaluated.

For the within-method agreement based on qualitative results and pattern, there was 100% qualitative agreement in all samples when read using Methods A and B. For Method C, 22 out of 25 specimens showed 100% qualitative agreement; homogenous low positive, nucleolar low positive, and nuclear membrane low positive specimens did not have complete qualitative agreement. Additionally, there was 100% pattern agreement within Methods A and B. For method C, there was 100% pattern agreement for 21 out of 25 specimens with outliers observed in homogenous low positive, nucleolar low positive and nuclear membrane low positive and nuclear membrane high positive specimens. The results are summarized in the following tables:

| Within-Method Qualitative Result Agreement | | | |
| --- | --- | --- | --- |
| Sample | Method A Agreement (95% CI) | Method B Agreement (95% CI) | Method C Agreement (95% CI) |
| Homogenous Low Positive | 100% (88.7-100%) | 100% (88.7-100%) | 76.7% (59.1-88.2%) |
| Homogenous Mid Positive | 100% (88.7-100%) | 100% (88.7-100%) | 100% (88.7-100%) |
| Homogenous High Positive | 100% (88.7-100%) | 100% (88.7-100%) | 100% (88.7-100%) |
| Speckled Low Positive | 100% (88.7-100%) | 100% (88.7-100%) | 100% (88.7-100%) |
| Speckled Mid Positive | 100% (88.7-100%) | 100% (88.7-100%) | 100% (88.7-100%) |
| Speckled High Positive | 100% (88.7-100%) | 100% (88.7-100%) | 100% (88.7-100%) |

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| Within-Method Qualitative Result Agreement | | | |
| --- | --- | --- | --- |
| Sample | Method A Agreement (95% CI) | Method B Agreement (95% CI) | Method C Agreement (95% CI) |
| Centromere
Low Positive | 100%
(88.7-100%) | 100%
(88.7-100%) | 100%
(88.7-100%) |
| Centromere
Mid Positive | 100%
(88.7-100%) | 100%
(88.7-100%) | 100%
(88.7-100%) |
| Centromere
High Positive | 100%
(88.7-100%) | 100%
(88.7-100%) | 100%
(88.7-100%) |
| Nucleolar
Low Positive | 100%
(88.7-100%) | 100%
(88.7-100%) | 96.7%
(83.3-99.4%) |
| Nucleolar
Mid Positive | 100%
(88.7-100%) | 100%
(88.7-100%) | 100%
(88.7-100%) |
| Nucleolar
High Positive | 100%
(88.7-100%) | 100%
(88.7-100%) | 100%
(88.7-100%) |
| Mitochondria
Low Positive | 100%
(88.7-100%) | 100%
(88.7-100%) | 100%
(88.7-100%) |
| Mitochondrial
Mid Positive | 100%
(88.7-100%) | 100%
(88.7-100%) | 100%
(88.7-100%) |
| Mitochondrial
High Positive | 100%
(88.7-100%) | 100%
(88.7-100%) | 100%
(88.7-100%) |
| Nuclear Membrane
Low Positive | 100%
(88.7-100%) | 100%
(88.7-100%) | 90%
(74.4-96.5%) |
| Nuclear Membrane
Mid Positive | 100%
(88.7-100%) | 100%
(88.7-100%) | 100%
(88.7-100%) |
| Nuclear Membrane
High Positive | 100%
(88.7-100%) | 100%
(88.7-100%) | 100%
(88.7-100%) |
| Nuclear Dots
Low Positive | 100%
(88.7-100%) | 100%
(88.7-100%) | 100%
(88.7-100%) |
| Nuclear Dots
Mid Positive | 100%
(88.7-100%) | 100%
(88.7-100%) | 100%
(88.7-100%) |
| Nuclear Dots
High Positive | 100%
(88.7-100%) | 100%
(88.7-100%) | 100%
(88.7-100%) |
| Ribosomal
Low Positive | 100%
(88.7-100%) | 100%
(88.7-100%) | 100%
(88.7-100%) |
| Ribosomal
Mid Positive | 100%
(88.7-100%) | 100%
(88.7-100%) | 100%
(88.7-100%) |
| Ribosomal
High Positive | 100%
(88.7-100%) | 100%
(88.7-100%) | 100%
(88.7-100%) |
| Negative | 100%
(88.7-100%) | 100%
(88.7-100%) | 100%
(88.7-100%) |

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K201956 - Page 9 of 26

| Within-Method Pattern Result Agreement | | | |
| --- | --- | --- | --- |
| Sample | Method A Agreement (95% CI) | Method B Agreement (95% CI) | Method C Agreement (95% CI) |
| Homogenous
Low Positive | 100%
(88.7-100%) | 100%
(88.7-100%) | 73.3%
(55.6-85.8) |
| Homogenous
Mid Positive | 100%
(88.7-100%) | 100%
(88.7-100%) | 100%
(88.7-100%) |
| Homogenous
High Positive | 100%
(88.7-100%) | 100%
(88.7-100%) | 100%
(88.7-100%) |
| Speckled
Low Positive | 100%
(88.7-100%) | 100%
(88.7-100%) | 100%
(88.7-100%) |
| Speckled
Mid Positive | 100%
(88.7-100%) | 100%
(88.7-100%) | 100%
(88.7-100%) |
| Speckled
High Positive | 100%
(88.7-100%) | 100%
(88.7-100%) | 100%
(88.7-100%) |
| Centromere
Low Positive | 100%
(88.7-100%) | 100%
(88.7-100%) | 100%
(88.7-100%) |
| Centromere
Mid Positive | 100%
(88.7-100%) | 100%
(88.7-100%) | 100%
(88.7-100%) |
| Centromere
High Positive | 100%
(88.7-100%) | 100%
(88.7-100%) | 100%
(88.7-100%) |
| Nucleolar
Low Positive | 100%
(88.7-100%) | 100%
(88.7-100%) | 96.7%
(83.3-99.4%) |
| Nucleolar
Mid Positive | 100%
(88.7-100%) | 100%
(88.7-100%) | 100%
(88.7-100%) |
| Nucleolar
High Positive | 100%
(88.7-100%) | 100%
(88.7-100%) | 100%
(88.7-100%) |
| Mitochondria
Low Positive | 100%
(88.7-100%) | 100%
(88.7-100%) | 100%
(88.7-100%) |
| Mitochondrial
Mid Positive | 100%
(88.7-100%) | 100%
(88.7-100%) | 100%
(88.7-100%) |
| Mitochondrial
High Positive | 100%
(88.7-100%) | 100%
(88.7-100%) | 100%
(88.7-100%) |
| Nuclear Membrane
Low Positive | 100%
(88.7-100%) | 100%
(88.7-100%) | 86.67%
(70.3-94.7%) |
| Nuclear Membrane
Mid Positive | 100%
(88.7-100%) | 100%
(88.7-100%) | 100%
(88.7-100%) |
| Nuclear Membrane
High Positive | 100%
(88.7-100%) | 100%
(88.7-100%) | 96.7%
(83.3-99.4%) |
| Nuclear Dots
Low Positive | 100%
(88.7-100%) | 100%
(88.7-100%) | 100%
(88.7-100%) |
| Nuclear Dots
Mid Positive | 100%
(88.7-100%) | 100%
(88.7-100%) | 100%
(88.7-100%) |
| Nuclear Dots
High Positive | 100%
(88.7-100%) | 100%
(88.7-100%) | 100%
(88.7-100%) |

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K201956 - Page 10 of 26

| Within-Method Pattern Result Agreement | | | |
| --- | --- | --- | --- |
| Sample | Method A Agreement (95% CI) | Method B Agreement (95% CI) | Method C Agreement (95% CI) |
| Ribosomal Low Positive | 100% (88.7-100%) | 100% (88.7-100%) | 100% (88.7-100%) |
| Ribosomal Mid Positive | 100% (88.7-100%) | 100% (88.7-100%) | 100% (88.7-100%) |
| Ribosomal High Positive | 100% (88.7-100%) | 100% (88.7-100%) | 100% (88.7-100%) |

For the between-method agreement based on qualitative results and pattern, there was 100% qualitative and pattern agreement between Method A versus Method B. However, only 22 out of 25 specimens showed 100% qualitative in the comparisons between Method B vs Method C and Method A vs Method C; the discrepant samples were the homogenous low positive, nucleolar low positive and nuclear membrane low positive specimens. Additionally, only 21 out of 25 specimens showed 100% pattern agreement in the comparisons between Method B vs Method C and Method A vs Method C; the discrepant samples were the homogenous low positive, nucleolar low positive and nuclear membrane low positive and nuclear membrane high positive specimens. The results are summarized in the following tables:

| Between-Method Qualitative Result Agreement | | | |
| --- | --- | --- | --- |
| Sample | Method A vs Method B | Method A vs Method C | Method B vs Method C |
| | Agreement (95% CI) | Agreement (95% CI) | Agreement (95% CI) |
| Homogenous Low Positive | 100% (88.7-100%) | 76.67% (59.1-88.2%) | 76.67% (59.1-88.2) |
| Homogenous Mid Positive | 100% (88.7-100%) | 100% (88.7-100%) | 100% (88.7-100%) |
| Homogenous High Positive | 100% (88.7-100%) | 100% (88.7-100%) | 100% (88.7-100%) |
| Speckled Low Positive | 100% (88.7-100%) | 100% (88.7-100%) | 100% (88.7-100%) |
| Speckled Mid Positive | 100% (88.7-100%) | 100% (88.7-100%) | 100% (88.7-100%) |
| Speckled High Positive | 100% (88.7-100%) | 100% (88.7-100%) | 100% (88.7-100%) |
| Centromere Low Positive | 100% (88.7-100%) | 100% (88.7-100%) | 100% (88.7-100%) |
| Centromere Mid Positive | 100% (88.7-100%) | 100% (88.7-100%) | 100% (88.7-100%) |
| Centromere High Positive | 100% (88.7-100%) | 100% (88.7-100%) | 100% (88.7-100%) |

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K201956 - Page 11 of 26

| Between-Method Qualitative Result Agreement | | | |
| --- | --- | --- | --- |
| Sample | Method A vs Method B | Method A vs Method C | Method B vs Method C |
| | Agreement (95% CI) | Agreement (95% CI) | Agreement (95% CI) |
| Nucleolar | 100% | 96.67% | 96.67% |
| Low Positive | (88.7-100%) | (83.3-99.4) | (83.3-99.4) |
| Nucleolar | 100% | 100% | 100% |
| Mid Positive | (88.7-100%) | (88.7-100%) | (88.7-100%) |
| Nucleolar | 100% | 100% | 100% |
| High Positive | (88.7-100%) | (88.7-100%) | (88.7-100%) |
| Mitochondria | 100% | 100% | 100% |
| Low Positive | (88.7-100%) | (88.7-100%) | (88.7-100%) |
| Mitochondrial | 100% | 100% | 100% |
| Mid Positive | (88.7-100%) | (88.7-100%) | (88.7-100%) |
| Mitochondrial | 100% | 100% | 100% |
| High Positive | (88.7-100%) | (88.7-100%) | (88.7-100%) |
| Nuclear Membrane | 100% | 90% | 90% |
| Low Positive | (88.7-100%) | (74.4-96.5%) | (74.4-96.5%) |
| Nuclear Membrane | 100% | 100% | 100% |
| Mid Positive | (88.7-100%) | (88.7-100%) | (88.7-100%) |
| Nuclear Membrane | 100% | 100% | 100% |
| High Positive | (88.7-100%) | (88.7-100%) | (88.7-100%) |
| Nuclear Dots | 100% | 100% | 100% |
| Low Positive | (88.7-100%) | (88.7-100%) | (88.7-100%) |
| Nuclear Dots | 100% | 100% | 100% |
| Mid Positive | (88.7-100%) | (88.7-100%) | (88.7-100%) |
| Nuclear Dots | 100% | 100% | 100% |
| High Positive | (88.7-100%) | (88.7-100%) | (88.7-100%) |
| Ribosomal | 100% | 100% | 100% |
| Low Positive | (88.7-100%) | (88.7-100%) | (88.7-100%) |
| Ribosomal | 100% | 100% | 100% |
| Mid Positive | (88.7-100%) | (88.7-100%) | (88.7-100%) |
| Ribosomal | 100% | 100% | 100% |
| High Positive | (88.7-100%) | (88.7-100%) | (88.7-100%) |
| Between-Method Pattern Result Agreement | | | |
| --- | --- | --- | --- |
| Sample | Method A vs Method B | Method A vs Method C | Method B vs Method C |
| | Agreement (95% CI) | Agreement (95% CI) | Agreement (95% CI) |
| Homogenous | 100% | 73.3% | 73.3% |
| Low Positive | (88.7-100%) | (55.6-85.9%) | (55.6-85.9%) |
| Homogenous | 100% | 100% | 100% |
| Mid Positive | (88.7-100%) | (88.7-100%) | (88.7-100%) |
| Homogenous | 100% | 100% | 100% |
| High Positive | (88.7-100%) | (88.7-100%) | (88.7-100%) |

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K201956 - Page 12 of 26

| Between-Method Pattern Result Agreement | | | |
| --- | --- | --- | --- |
| Sample | Method A vs Method B
Agreement (95% CI) | Method A vs Method C
Agreement (95% CI) | Method B vs Method C
Agreement (95% CI) |
| Speckled Low Positive | 100% (88.7-100%) | 100% (88.7-100%) | 100% (88.7-100%) |
| Speckled Mid Positive | 100% (88.7-100%) | 100% (88.7-100%) | 100% (88.7-100%) |
| Speckled High Positive | 100% (88.7-100%) | 100% (88.7-100%) | 100% (88.7-100%) |
| Centromere Low Positive | 100% (88.7-100%) | 100% (88.7-100%) | 100% (88.7-100%) |
| Centromere Mid Positive | 100% (88.7-100%) | 100% (88.7-100%) | 100% (88.7-100%) |
| Centromere High Positive | 100% (88.7-100%) | 100% (88.7-100%) | 100% (88.7-100%) |
| Nucleolar Low Positive | 100% (88.7-100%) | 96.7% (83.3-99.4%) | 96.7% (83.3-99.4%) |
| Nucleolar Mid Positive | 100% (88.7-100%) | 100% (88.7-100%) | 100% (88.7-100%) |
| Nucleolar High Positive | 100% (88.7-100%) | 100% (88.7-100%) | 100% (88.7-100%) |
| Mitochondria Low Positive | 100% (88.7-100%) | 100% (88.7-100%) | 100% (88.7-100%) |
| Mitochondrial Mid Positive | 100% (88.7-100%) | 100% (88.7-100%) | 100% (88.7-100%) |
| Mitochondrial High Positive | 100% (88.7-100%) | 100% (88.7-100%) | 100% (88.7-100%) |
| Nuclear Membrane Low Positive | 100% (88.7-100%) | 86.7% (70.3-94.7%) | 86.7% (70.3-94.7%) |
| Nuclear Membrane Mid Positive | 100% (88.7-100%) | 100% (88.7-100%) | 100% (88.7-100%) |
| Nuclear Membrane High Positive | 100% (88.7-100%) | 96.7% (83.3-99.4%) | 96.7% (83.3-99.4%) |
| Nuclear Dots Low Positive | 100% (88.7-100%) | 100% (88.7-100%) | 100% (88.7-100%) |
| Nuclear Dots Mid Positive | 100% (88.7-100%) | 100% (88.7-100%) | 100% (88.7-100%) |
| Nuclear Dots High Positive | 100% (88.7-100%) | 100% (88.7-100%) | 100% (88.7-100%) |
| Ribosomal Low Positive | 100% (88.7-100%) | 100% (88.7-100%) | 100% (88.7-100%) |
| Ribosomal Mid Positive | 100% (88.7-100%) | 100% (88.7-100%) | 100% (88.7-100%) |
| Ribosomal High Positive | 100% (88.7-100%) | 100% (88.7-100%) | 100% (88.7-100%) |

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k201956 - Page 13 of 26

b. Reproducibility – 5-Day Site-to-Site Reproducibility Study

A low positive serum sample (~1:40 endpoint), a mid-positive serum sample (~1:160 to 1:320 endpoint) and a strong positive serum sample (>1:640 endpoint) were identified for each of the eight ANA patterns reported by dIFine. One ANA negative specimen was also included bringing the group of specimens to a total of 25. These 25 specimens were assayed (1:40 screening dilution) in triplicate, two times per day on five different days at three different sites/laboratories, producing 30 results per sample, per location. At each site, Method A and Method B were also interpreted by two separate laboratory technicians. Therefore, for Method A and Method B, there were 30 assays of each of the 25 specimens but 60 interpretations of those 25 specimens. The slides were interpreted by all three methods for qualitative results and pattern determinations for all positive samples.

Qualitative Agreement:

Within Method:
Within method qualitative agreement for Method A and Method B was 100% for all three sites and all technicians. For Method C, there was 100% agreement for 24 out of 25 specimens at all sites and all technicians; one nuclear membrane low positive specimen was discrepant at Site 2.

Between Method:
There was 100% qualitative agreement for all 25 specimens between Method A versus Method B and Method A versus Method C. Notably, there was 100% qualitative agreement between Method B vs Method C for 24 out of 25 specimens with the nuclear membrane low positive specimen being discrepant at Site 2.

Pattern Agreement:

Within Method:
Within method pattern agreement for Method A and Method B was 100% for all three sites and all technicians. For Method C, there was 100% agreement for 24 out of 25 specimens with centromere mid specimen as the only discrepant result.

Between Method:
There was 100% pattern agreement for all 25 specimens between Method A vs B for all sites and all technicians. For Method A vs Method C, there were two discrepant samples: a centromere mid specimen at Site 1 and a nuclear membrane low specimen at Site 2. Similarly, for Method B vs Method C, there were two discrepant samples: a centromere mid specimen at Site 1 and a nuclear membrane low specimen at Site 2.

c. Lot-to-Lot Precision

The lot-to-lot precision of the qualitative and pattern results was evaluated in a 5-Day study using three lots of Zeus IFA ANA Hep-2 Test. A mid positive serum sample (~1:160 to 1:320 endpoint) and a strong positive sample (>1:640 endpoint) were identified for each of the eight ANA patterns. Nine ANA negative specimens were also included bringing the group of specimens to a total of 25 samples. The 16 positive

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samples were serially titered through endpoint, assayed on three separate lots of Zeus IFA ANA Hep-2 Test system and then scanned by Zeus dIFine. The nine negative samples were assayed at a 1:40 screening dilution on three separate lots of Zeus IFA ANA Hep-2 Test system and then scanned by Zeus dIFine. The slides were interpreted by all three methods for qualitative results and pattern determinations.

**Qualitative Agreement:** There was 100% agreement in the qualitative results at the screening dilution of all 25 specimens across all three kit lots, and there was 100% agreement across all three interpretation methods regardless of reagent kit lot.

**Endpoint Titer Agreement:** All 16 positive specimens resulted in endpoint titers +/- one dilution regardless of reagent kit lot or method interpretation.

**Pattern Agreement:** For lot 1 and lot 2, there was 100% pattern agreement for each specimen by all three interpretation methods. For lot 3, there was 100% pattern agreement for each specimen between Method A and Method B, with one homogenous mid positive specimen as the only discrepant specimen when comparing Method C versus Methods A and B.

## 2. Linearity:

A strong positive serum sample was identified for each of the eight ANA patterns. Each of the samples was serially diluted from a 1:40 titer to 1:20,480 titer then qualitatively interpreted as positive or negative by all three methods as noted above. The endpoint titer was considered as the last positive well within a titration series. In general, the fluorescent intensity decreased (e.g., +4 to +1) with an increase in titer for the samples. Patterns did not change when the samples were diluted; the same patterns were recovered in every dilution until negative.

The endpoint titers across samples for each method for each of eight samples with eight ANA patterns are shown in the table below:

| Endpoint Titers across Samples and Methods | | | |
| --- | --- | --- | --- |
| Sample | Method A | Method B | Method C |
| Homogenous | 1:1280 | 1:1280 | 1:1280 |
| Speckled | 1:5120 | 1:5120 | 1:5120 |
| Centromere | 1:5120 | 1:5120 | 1:5120 |
| Nucleolar | 1:2560 | 1:5120 | 1:2560 |
| Nuclear Dots | 1:640 | 1:640 | 1:640 |
| Nuclear Membrane | 1:2560 | 1:2560 | 1:2560 |
| Cytoplasmic (Ribosomal) | 1:320 | 1:320 | 1:320 |
| Cytoplasmic (Mitochondrial) | 1:10240 | 1:10240 | 1:10240 |

K201956 - Page 14 of 26

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In all samples the pattern called was as expected and in agreement regardless of the method of interpretation. Likewise, the endpoint determination was within one dilution for all of the determinations regardless of the method of interpretation.

# 3. Analytical Specificity/Interference:

# a. Interference studies:

The interference study was performed according to CLSI EP07-A2 to determine the effect of various endogenous and exogenous substances on the ZEUS IFA ANA HEp-2 Test System in combination with ZEUS dIFine. A mid-positive sample (~1:160 to 1:320 endpoint) and a strong positive sample (≥1:640 endpoint) were identified for each of the eight ANA patterns. One ANA negative specimen was also included bringing the group of specimens to a total of 17. These 17 specimens were spiked with two different concentrations (low spike and high spike) of 12 different possible interferents as outlined in the table below. All specimens were evaluated on the ZEUS IFA ANA HEp-2 Test System and interpreted by all three methods. For methods A and B, neither the qualitative agreement nor the resulting pattern was affected by the addition of the possible interferents. Method C yielded one uncertain result in a low positive nuclear membrane sample when spiked with Albumin.

A second interference study was conducted using a low positive sample (approximate endpoint titer 1:40/1:80), for each of the eight ANA patterns along with one negative serum sample tested with some additional potential interferents. Exogenous and endogenous interferants were added at high and low concentrations to each of the serum samples listed. Each of the nine serum samples listed above were assayed at a 1:40 sample dilution using the ZEUS IFA ANA HEp-2 Test System, then scanned by ZEUS dIFine. Qualitative results (i.e., Positive or Negative) were determined via Methods A, B, and C for all samples.

| Endogenous and Exogenous Interferants Tested | | | |
| --- | --- | --- | --- |
| Endogenous Interfering Substance | Maximum Concentration | Exogenous Interfering Substance | Maximum Concentration |
| Hemoglobin | 200 mg/mL | Prednisone | 0.000099 mg/mL |
| Bilirubin | 0.15 mg/mL | Cyclophosphamide | 0.549 mg/mL |
| Triglycerides | 2.5 mg/mL | Ibuprofen | 0.219 mg/mL |
| Cholesterol | 2.2 mg/mL | Hydroxychloroquine | 0.24 mg/mL |
| Rheumatoid Factor | 400 IU/mL | Simvastatin | 0.000083 mg/mL |
| Intralipids | 20 mg/mL | Azathioprine | 0.00258 mg/mL |
| Albumin | 52 mg/mL | Diltiazem | 0.0009 mg/mL |
| | | Mycophenolate Mofetil | 0.048 mg/mL |
| | | Rituximab | 2 mg/mL |
| | | Belimumab | 8 mg/mL |

Neither the qualitative agreement nor the resulting pattern was affected by the addition of the possible interferents at the concentrations listed in the table, regardless of the method of interpretation.

K201956 - Page 15 of 26

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b. Cross-reactivity

The analytical cross-reactivity of the assay was evaluated using 23 International Consensus on Antinuclear Antibody (ANA) Patterns (ICAP) reference samples tested at a 1:40 dilution with all three interpretation methods. All qualitative results are in line with the characterization by ICAP and did not indicate cross-reactivity to ANA.

4. Assay Reportable Range:

Not applicable

5. Traceability, Stability, Expected Values (Controls, Calibrators, or Methods):

a. Traceability

There is no recognized standard for anti-nuclear antibodies.

b. Stability

i). Open Reagent Stability

The slides must be used the same day as they are opened. All other ready-to-use reagents, except PBS, may be used until their stated expiration date.

ii). Unopened Reagent Stability

The reagents for this assay are the same as those in the predicate device. The real-time stability studies support shelf-life claim of 24 months when stored at 2–8°C.

iii). Specimen Stability

The real-time sample stability data supports sample storage at room temperature (20–25°C) for no longer than eight hours. If testing is not performed within eight hours, sera may be stored between 2–8°C, for no longer than 48 hours. If delay in testing is anticipated, test sera must be stored at –20°C or lower.

6. Detection Limit:

Not applicable

7. Assay Cut-Off:

Please refer to K781516. Zeus recommends a screening dilution of 1:40 and any titers less than 1:40 are considered negative.

8. Accuracy (Instrument):

Not applicable

K201956 - Page 16 of 26

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9. Carry-Over:
Not applicable

B Comparison Studies:

1. Method Comparison with Predicate Device:

The method comparison study was performed using clinical samples (described in Section C.1) at three independent laboratories in the U.S. At Sites 1 and 3, 392 samples were tested; at Site 2, 350 samples were tested. Two technicians tested each sample at each site. Method A is considered as the predicate.

a. Qualitative comparisons:

The qualitative agreement between the methods was determined by testing a 1:40 sample dilution of each sample with the ZEUS IFA ANA HEp-2 Test System then read using all three methods. Method A and Method B readings were read using two different laboratory technicians. The positive samples were titered to endpoint and the pattern was determined. Positive percent agreement (PPA) and negative percent agreement (NPA) values were calculated, with 95% confidence intervals.

i. Method A vs Method B Qualitative Comparison

| Qualitative Agreement between Method A and Method B | | | | |
| --- | --- | --- | --- | --- |
| Method A vs Method B | | Positive Sample Agreement (95% CI) | Negative Sample Agreement (95% CI) | Total Sample Agreement (95% CI) |
| Site 1 | Tech 1 | 95.6%
(152/159)
(91.2-97.9%) | 95.7%
(223/233)
(92.3-97.7%) | 95.7%
(375/392)
(93.2-97.3%) |
| | Tech 2 | 94.9%
(168/177)
(90.6-97.3%) | 98.6%
(212/215)
(95.9-99.5%) | 96.9%
(380/392)
(94.7-98.2%) |
| Site 2 | Tech 1 | 99.2%
(124/125)
(95.6-99.9%) | 93.8%
(211/225)
(89.8-96.3%) | 95.7%
(335/350)
(93.1-97.4%) |
| | Tech 2 | 98.6%
(137/139)
(94.9-99.6%) | 97.6%
(206/211)
(94.6-98.9%) | 98.0%
(343/350)
(95.9-99.0%) |
| Site 3 | Tech 1 | 95.5%
(148/155)
(90.9-97.8%) | 98.7%
(234/237)
(96.4-99.6%) | 97.5%
(382/392)
(95.4-98.6%) |
| | Tech 2 | 95.9%
(162/169)
(91.7-97.9%) | 97.8%
(218/223)
(94.9-99.0%) | 96.9%
(380/392)
(94.7-98.2%) |

K201956 - Page 17 of 26

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| Combined Qualitative Agreement for Method A and Method B | | | | |
| --- | --- | --- | --- | --- |
| | Method A | | Total | |
| | | Positive | | Negative |
| Method B | Positive | 891 | 40 | 931 |
| | Negative | 33 | 1304 | 1337 |
| Total | | 924 | 1344 | 2268 |
| PPA: 96.4% (891/924) (95% CI: 95.0%; 97.5%) NPA: 97.0% (1304/1344) (95% CI: 96.0%; 97.8%) Overall Agreement: 96.8% (2195/2268) (95% CI: 96.0%; 97.4%) | | | | |

## ii. Method A vs Method C Qualitative Comparison

Since Method C can yield an uncertain (UNC) result in addition to a positive or negative qualitative result, the agreement between methods were calculated using the UNC samples considered positive and then considered negative:

| Qualitative Agreement between Method A and Method C (When UNC from Method C Considered as Positive) | | | | |
| --- | --- | --- | --- | --- |
| Method A vs Method C | | Positive Sample Agreement (95% CI) | Negative Sample Agreement (95% CI) | Total Sample Agreement (95% CI) |
| Site 1 | Tech 1 | 97.5% (155/159) (93.7-99.0%) | 80.3% (187/233) (74.7-84.9%) | 87.2% (342/392) (83.6-90.2%) |
| | Tech 2 | 98.3% (174/177) (95.1-99.4%) | 87.4% (188/215) (82.4-91.2%) | 92.4% (362/392) (89.3-94.6%) |
| Site 2 | Tech 1 | 100% (125/125) (97.0-100.0%) | 85.3% (192/225) (80.1-89.4%) | 90.6% (317/350) (87.1-93.2%) |
| | Tech 2 | 100% (139/139) (97.3-100.0%) | 91.0% (192/211) (86.4-94.2%) | 94.6% (331/350) (91.7-96.5%) |
| Site 3 | Tech 1 | 100% (155/155) (97.6-100.0%) | 87% (206/237) (82.0-90.7%) | 92.1% (361/392) (88.9-94.4%) |
| | Tech 2 | 98.8% (167/169) (95.8-99.7%) | 91.9% (205/223) (87.6-94.8%) | 94.9% (372/392) (92.3-96.7%) |

K201956 - Page 18 of 26

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| Combined Qualitative Agreement for Method A and Method C - When UNC from Method C Considered as Positive | | | | |
| --- | --- | --- | --- | --- |
| | Method A | | Total | |
| | | Positive | | Negative |
| Method C | Positive | 915 | 174 | 1089 |
| | Negative | 9 | 1170 | 1179 |
| Total | | 924 | 1344 | 2268 |
| PPA: 99.0% (915/924) (95% CI: 98.4%; 99.6%) NPA: 87.1% (1170/1344) (95% CI: 85.2%; 88.7%) Overall Agreement: 91.9% (2085/2268) (95% CI: 90.7%; 93.0%) | | | | |
| Qualitative Agreement between Method A and Method C (When UNC from Method C Considered as Negative) | | | | |
| --- | --- | --- | --- | --- |
| Method A vs Method C | | Positive Sample Agreement (95% CI) | Negative Sample Agreement (95% CI) | Total Sample Agreement (95% CI) |
| Site 1 | Tech 1 | 93.1% (148/159) (88.0-96.1%) | 94.0% (219/233) (90.2-96.4%) | 93.6% (367/392) (90.8-96.5%) |
| | Tech 2 | 90.4% (160/177) (85.2-93.9%) | 99.1% (213/215) (96.7-99.7%) | 95.2% (373/392) (92.6-96.9%) |
| Site 2 | Tech 1 | 99.2% (124/125) (95.1-99.9%) | 94.7% (213/225) (90.9-96.9%) | 96.3% (337/350) (93.8-97.8%) |
| | Tech 2 | 95.0% (132/139) (89.9-97.5%) | 98.1% (207/211) (95.2-99.3%) | 96.9% (339/350) (94.5-98.2%) |
| Site 3 | Tech 1 | 96.1% (149/155) (91.8-98.2%) | 97.1% (230/237) (94.4-98.1%) | 96.7% (379/392) (94.4-98.1%) |
| | Tech 2 | 90.5% (153/169) (85.2-94.1%) | 98.7% (220/223) (96.1-99.5%) | 95.2% (373/392) (92.6-96.9%) |

K201956 - Page 19 of 26

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| Combined Qualitative Agreement for Method A and Method C - (When UNC from Method C Considered as Negative) | | | | |
| --- | --- | --- | --- | --- |
| | Method A | | Total | |
| | | Positive | | Negative |
| Method C | Positive | 866 | 42 | 908 |
| | Negative | 58 | 1302 | 1360 |
| Total | | 924 | 1344 | 2268 |
| PPA: 93.7% (866/924) (95% CI: 92.0%; 95.1%) NPA: 96.9% (1302/1344) (95% CI: 95.8%; 97.7%) Overall Agreement: 95.6% (2168/2268) (95% CI: 94.7%; 96.4%) | | | | |

iii. Method B vs Method C Qualitative Comparison

Similar to the approach as presented in tables 10 through 13 above, UNC calls in Method C were counted towards both negatives as well as positives to determine the qualitative agreement between Method B versus Method C. The positive agreement, negative agreement and the total agreement between Method B versus Method C at each site and each technician as well combined for all sites and all technicians was similar to the results observed for Method A versus Method C.

b. Pattern Agreement

Additionally, pattern determinations were correlated across different interpretation methods for each sample included in the study. For the purpose of pattern recognition, samples that had no pattern (i.e., 'negative') or samples that yielded uncertain results (i.e., 'UNC') were included in the pattern agreement analyses between the different interpretation methods and are presented in the table below. Low titer samples, samples containing mixed patterns, and/or samples containing esoteric patterns (e.g., Golgi-like and centromere protein-F (CENP)-like patterns), were most likely to yield discrepant pattern results across interpretation methods and testing sites.

| Pattern Agreement Between Methods at each Site and each Technician | | | | |
| --- | --- | --- | --- | --- |
| | Method A vs Method B | Method A vs Method C | Method B vs Method C | |
| | | % Agreement (95% CI) | % Agreement (95% CI) | % Agreement (95% CI) |
| Site 1 | Tech 1 | 98.5% (386/392) (96.7-99.3%) | 84.2% (330/392) (80.2-87.5%) | 83.9% (329/392) (80.0-87.2%) |
| | Tech 2 | 97.7% (383/392) (95.7-98.8%) | 85.0% (333/392) (81.1-88.2%) | 85.2% (334/392) (81.4-88.4%) |

K201956 - Page 20 of 26

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| Pattern Agreement Between Methods at each Site and each Technician | | | | |
| --- | --- | --- | --- | --- |
| | Method A vs Method B | Method A vs Method C | Method B vs Method C | |
| | | % Agreement (95% CI) | % Agreement (95% CI) | % Agreement (95% CI) |
| Site 2 | Tech 1 | 96.9%
(339/350)
(94.5-98.2%) | 86.0%
(301/350)
(82.0-89.3%) | 86.9%
(304/350)
(82.9-90.0%) |
| | Tech 2 | 97.7%
(342/350)
(95.6-98.8%) | 86.6%
(303/350)
(82.6-89.8%) | 87.1%
(305/350)
(83.2-90.3%) |
| Site 3 | Tech 1 | 98.47%
(386/392)
(96.7-99.3%) | 88.3%
(346/392)
(84.7-91.1%) | 88.5%
(347/392)
(85.0-91.3%) |
| | Tech 2 | 98.2%
(385/392)
(96.4-99.1%) | 88.0%
(345/392)
(84.4-90.9%) | 87.5%
(343/392)
(83.9-90.4%) |
| Combined Pattern Agreement for all Sites and all Technicians | | | |
| --- | --- | --- | --- |
| | Agreement #/Total Samples | % Agreement | 95% CI |
| Method A vs Method B | 2221/2268 | 98.0% | 97.3-98.4% |
| Method A vs Method C | 1958/2268 | 86.3% | 84.9-87.7% |
| Method B vs Method C | 1962/2268 | 86.5% | 85.0-87.9% |

c. **End-point Titer Agreement**

Samples that were positive at each site were titrated to endpoint and interpreted by all three methods. Considering there were two technicians at each site to read by both Method A and Method B, this resulted in 886 comparisons in endpoint determinations between Method A versus Method B, Method A versus Method C and Method B versus Method C.

| Combined Endpoint Titer Agreement between Methods for all Technicians and all Sites | | |
| --- | --- | --- |
| Interpretation | +/- one dilution/Total samples | %Agreement (95% CI) |
| Method A vs Method B | 880/886 | 99.3% (98.5 - 99.7%) |
| Method A vs Method C | 879/886 | 99.2% (98.4 - 99.6%) |
| Method B vs Method C | 880/886 | 99.3% (98.5 - 99.7%) |

2. **Matrix Comparison:**

Not applicable

K201956 - Page 21 of 26

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C Clinical Studies:

1. Clinical Sensitivity and Clinical Specificity:

Clinically characterized specimens, described below, were tested to determine the clinical sensitivity and specificity of the assay on all three methods. The samples were acquired from commercial sources, aliquoted into three samples, then tested at three separate clinical laboratories. At all sites, the same cohort of 190 samples associated with ANA-associated autoimmune diseases were tested, including systemic autoimmune rheumatic diseases (SARDs). At Site 1 and Site 3, 190 samples from diseases that usually are not associated with SARDs were tested; a smaller set of 160 samples was tested at Site 2. The samples were assayed at the screening 1:40 sample dilution then read by all three methods. At all three sites, Method A and Method B were interpreted by two different laboratory technicians. Samples positive at the 1:40 screening dilution were subsequently titered to determine the endpoint and pattern.

| Clinically Characterized Samples used in the Study | | | | |
| --- | --- | --- | --- | --- |
| ANA-associated diseases | n, All Sites | Non- ANA-associated diseases | n, Site 1 and Site 3 | n, Site 2 |
| SARDs: | | Other autoimmune diseases: | | |
| Systemic Lupus Erythematosus (SLE) | 40 | Celiac Disease | 22 | 10 |
| Sjögren's Syndrome (SS) | 30 | ANCA-associated Vasculitis | 28 | 10 |
| Scleroderma | 20 | Crohn's Disease | 10 | 10 |
| Autoimmune Myositis (AM) | 20 | Rheumatoid Arthritis | 30 | 30 |
| Mixed Connective Tissue Disease (MCTD) | 20 | Inflammatory Bowel Disease (IBD) | 10 | 10 |
| CREST | 20 | Autoimmune Thyroiditis | 30 | 30 |
| Other ANA-associated autoimmune diseases: | | Ulcerative Colitis | 10 | 10 |
| Autoimmune Hepatitis (AIH) | 20 | Other diseases: | | |
| Drug-induced Lupus (DIL) | 20 | Malignancy/cancer | 20 | 20 |
| | | Fibromyalgia | 10 | 10 |
| | | Infectious diseases | 10 | 10 |
| Total | 190 | Total | 190 | 160 |

The clinical sensitivity was calculated at each site on SLE separately, and on the SARDs plus other ANA-associated diseases (autoimmune liver hepatitis + Drug-induced Lupus). Specificity was calculated using the non-ANA-associated diseases described above.

The clinical sensitivity and specificity for each site and each technician is presented in the table below:

K201956 - Page 22 of 26

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| Clinical Performance at Site 1 | % Sensitivity (95% CI) | % Sensitivity (95% CI) | % Specificity (95% CI) | |
| --- | --- | --- | --- | --- |
| | | SLE (n = 40) | CTD + ANA-associated diseases (n = 190) | Non- ANA-associated diseases (n = 160) |
| Method A | Tech 1 | 52.5% (37.5-67.1%) | 53.2% (46.1-60.1%) | 75.8% (69.2-81.3%) |
| | Tech 2 | 57.5% (42.2-71.5%) | 60.0% (52.9-66.7%) | 73.2% (66.4-79.0%) |
| Method B | Tech 1 | 55.0% (39.8-69.3%) | 54.7% (47.6-61.6%) | 75.8% (69.2-81.3%) |
| | Tech 2 | 52.5% (37.5-67.1%) | 57.9% (50.8-64.7%) | 74.2% (67.6-79.9%) |
| Method C | dIFine | 55.0% (39.8-69.3%) | 57.4% (50.3-64.2%) | 67.4% (60.4-73.6%) |
| Clinical Performance at Site 2 | % Sensitivity (95% CI) | % Sensitivity (95% CI) | % Specificity (95% CI) | |
| --- | --- | --- | --- | --- |
| | | SLE (n = 40) | CTD + ANA-associated diseases (n = 190) | Non- ANA-associated diseases (n = 160) |
| Method A | Tech 1 | 50.0% (35.2-64.8%) | 51.6% (44.5-58.6%) | 83.1% (76.6-88.1%) |
| | Tech 2 | 55.0% (39.8-69.3%) | 56.3% (49.2-63.2%) | 80.0% (73.1-85.5%) |
| Method B | Tech 1 | 52.5% (37.5-67.1%) | 56.8% (49.7-63.7%) | 81.3% (74.5-86.5%) |
| | Tech 2 | 55.0% (39.8-69.3%) | 58.4% (51.3-65.2%) | 80.6% (73.8-86.0%) |
| Method C | dIFine | 52.5%(37.5-67.1%) | 55.8% (48.7-62.7%) | 74.4% (67.1-80.5%) |
| Clinical Performance at Site 3 | % Sensitivity (95% CI) | % Sensitivity (95% CI) | % Specificity (95% CI) | |
| --- | --- | --- | --- | --- |
| | | SLE (n = 40) | CTD + ANA-associated diseases (n = 190) | Non- ANA-associated diseases (n = 190) |
| Method A | Tech 1 | 47.5% (32.9-62.5%) | 53.2% (46.1-60.1%) | 76.8% (70.4-82.3%) |
| | Tech 2 | 55.0% (39.8-69.3%) | 57.4% (50.3-64.2%) | 74.2% (67.6-79.9%) |
| Method B | Tech 1 | 45.0% (30.7-60.2%) | 52.6% (45.6-59.6%) | 78.9% (72.6-84.1%) |
| | Tech 2 | 57.5% (42.2-71.5%) | 56.3% (49.2-63.2%) | 74.7% (68.1-80.4%) |
| Method C | dIFine | 55.0% (39.8-69.3%) | 54.2% (47.1-61.1%) | 67.5% (63.1-76.1%) |

2. Percent Positivity

Percent positivity rates in the various disease cohorts by three different interpretation methods at the three sites are listed below:

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| Percent Positivity for all Disease Types at Site 1 | | | | | |
| --- | --- | --- | --- | --- | --- |
| Diseases | Technician 1 | | Technician 2 | | |
| | Method A | Method B | Method A | Method B | Method C |
| ANA-associated Diseases | | | | | |
| SLE | 52.5% | 55.0% | 57.5% | 52.5% | 55.0% |
| Sjögren's Syndrome | 50.0% | 50.0% | 56.7% | 53.3% | 53.3% |
| Scleroderma | 75.0% | 70.0% | 80.0% | 80.0% | 80.0% |
| Autoimmune Myositis | 50.0% | 55.0% | 65.0% | 60.0% | 60.0% |
| MCTD | 40.0% | 40.0% | 50.0% | 55.0% | 50.0% |
| CREST | 80.0% | 80.0% | 80.0% | 80.0% | 80.0% |
| Autoimmune Hepatitis | 45.0% | 50.0% | 60.0% | 55.0% | 50.0% |
| Drug-Induces Lupus | 35.0% | 40.0% | 35.0% | 35.0% | 35.0% |
| Non-ANA associated Diseases | | | | | |
| Autoimmune Thyroiditis | 23.3% | 16.7% | 16.7% | 16.7% | 16.7% |
| Cancer | 15.0% | 15.0% | 20.0% | 20.0% | 15.0% |
| Celiac Disease | 45.5% | 45.5% | 45.5% | 45.5% | 45.5% |
| Crohn's Disease | 30.0% | 30.0% | 30.0% | 30.0% | 30.0% |
| Fibromyalgia | 30.0% | 40.0% | 50.0% | 40.0% | 40.0% |
| Infectious Disease | 5.0% | 5.0% | 5.0% | 5.0% | 5.0% |
| Inflammatory Bowel Disease | 40.0% | 30.0% | 40.0% | 20.0% | 20.0% |
| Rheumatoid Arthritis | 16.7% | 23.3% | 23.3% | 23.3% | 16.7% |
| Ulcerative Colitis | 30.0% | 30.0% | 50.0% | 60.0% | 40.0% |
| Vasculitis | 17.9% | 14.3% | 14.3% | 14.3% | 14.3% |
| Percent Positivity for all Disease Types at Site 2 | | | | | |
| --- | --- | --- | --- | --- | --- |
| Diseases | Technician 1 | | Technician 2 | | |
| | Method A | Method B | Method A | Method B | Method C |
| ANA-associated Diseases | | | | | |
| SLE | 50.0% | 52.5% | 55.0% | 55.0% | 52.5% |
| Sjögren's Syndrome | 53.3% | 53.3% | 56.7% | 56.7% | 53.3% |
| Scleroderma | 75.0% | 80.0% | 75.0% | 80.0% | 80.0% |
| Autoimmune Myositis | 55.0% | 65.0% | 65.0% | 70.0% | 60.0% |
| MCTD | 30.0% | 45.0% | 35.0% | 45.0% | 45.0% |
| CREST | 75.0% | 80.0% | 80.0% | 80.0% | 75.0% |
| Autoimmune Hepatitis | 40.0% | 45.0% | 45.0% | 45.0% | 45.0% |
| Drug-Induces Lupus | 35.0% | 40.0% | 40.0% | 40.0% | 40.0% |

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| Percent Positivity for all Disease Types at Site 2 | | | | | |
| --- | --- | --- | --- | --- | --- |
| Diseases | Technician 1 | | Technician 2 | | |
| | Method A | Method B | Method A | Method B | Method C |
| Non-ANA associated Diseases | | | | | |
| Autoimmune Thyroiditis | 13.3% | 16.7% | 16.7% | 16.7% | 16.7% |
| Cancer | 10.0% | 15.0% | 15.0% | 15.0% | 15.0% |
| Celiac Disease | 20.0% | 30.0% | 20.0% | 30.0% | 30.0% |
| Crohn's Disease | 30.0% | 30.0% | 30.0% | 30.0% | 30.0% |
| Fibromyalgia | 40.0% | 40.0% | 40.0% | 40.0% | 40.0% |
| Infectious Disease | 5.0% | 5.0% | 5.0% | 5.0% | 5.0% |
| Inflammatory Bowel Disease | 20.0% | 20.0% | 20.0% | 20.0% | 20.0% |
| Rheumatoid Arthritis | 16.7% | 16.7% | 16.7% | 16.7% | 16.7% |
| Ulcerative Colitis | 40.0% | 40.0% | 60.0% | 40.0% | 40.0% |
| Vasculitis | 0.0% | 0.0% | 10.0% | 10.0% | 0.0% |
| Percent Positivity for all Disease Types at Site 3 | | | | | |
| --- | --- | --- | --- | --- | --- |
| Diseases | Technician A | | Technician B | | |
| | Method A | Method B | Method A | Method B | Method C |
| ANA-associated Diseases | | | | | |
| SLE | 47.5% | 45.0% | 55.0% | 57.5% | 52.5% |
| Sjögren's Syndrome | 46.7% | 50.0% | 60.0% | 53.3% | 50.0% |
| Scleroderma | 75.0% | 75.0% | 80.0% | 75.0% | 75.0% |
| Autoimmune Myositis | 60.0% | 60.0% | 60.0% | 60.0% | 60.0% |
| MCTD | 45.0% | 45.0% | 45.0% | 45.0% | 45.0% |
| CREST | 80.0% | 80.0% | 75.0% | 75.0% | 75.0% |
| Autoimmune Hepatitis | 45.0% | 40.0% | 50.0% | 50.0% | 45.0% |
| Drug-Induces Lupus | 35.0% | 35.0% | 35.0% | 35.0% | 35.0% |
| Non-ANA associated Diseases | | | | | |
| Autoimmune Thyroiditis | 16.7% | 13.3% | 20.0% | 16.7% | 16.7% |
| Cancer | 20.0% | 20.0% | 20.0% | 25.0% | 15.0% |
| Celiac Disease | 45.5% | 45.5% | 45.5% | 45.5% | 45.5% |
| Crohn's Disease | 30.0% | 20.0% | 30.0% | 30.0% | 30.0% |
| Fibromyalgia | 40.0% | 40.0% | 50.0% | 50.0% | 40.0% |
| Infectious Disease | 5.0% | 5.0% | 5.0% | 5.0% | 5.0% |
| Inflammatory Bowel Disease | 30.0% | 30.0% | 30.0% | 30.0% | 20.0% |

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| Percent Positivity for all Disease Types at Site 3 | | | | | |
| --- | --- | --- | --- | --- | --- |
| Diseases | Technician A | | Technician B | | |
| | Method A | Method B | Method A | Method B | Method C |
| Rheumatoid Arthritis | 23.3% | 16.7% | 26.7% | 20.0% | 16.7% |
| Ulcerative Colitis | 40.0% | 40.0% | 60.0% | 60.0% | 40.0% |
| Vasculitis | 10.7% | 10.7% | 10.7% | 14.3% | 14.3% |

3. Other Clinical Supportive Data (When 1. and 2. Are Not Applicable):

Not applicable

D Clinical Cut-Off:

Not applicable

E Expected Values/Reference Range:

One hundred and eighty serum samples were acquired from apparently healthy donors. Samples were collected from donors within the United States. Each sample was assayed at the 1:40 screening titer using the ZEUS IFA ANA HEp-2 Test System, then scanned by ZEUS dIFine and the qualitative results (i.e., Positive, Negative or Uncertain) were determined via Methods A, B, and C. Percent positivity was determined for the 1:40 screening data. The results for the reference range are summarized below in the following table:

| Reference Range Determination (N=180) | | | | | | |
| --- | --- | --- | --- | --- | --- | --- |
| Method | Number of Positives | % Positives | Number of Negatives | % Negatives | Number of Uncertain | % Uncertain |
| A | 19 | 10.6% | 161 | 89.4% | NA | NA |
| B | 19 | 10.6% | 161 | 89.4% | NA | NA |
| C | 14 | 7.8% | 152 | 84.4% | 14 | 7.8% |

F Other Supportive Instrument Performance Characteristics Data:

Not Applicable

VIII Proposed Labeling:

The labeling supports the finding of substantial equivalence for this device.

IX Conclusion:

The submitted information in this premarket notification is complete and supports a substantial equivalence decision.

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