NOVA Lite DAPI ANA Kit

K150155 · Inova Diagnostics, Inc. · DHN · Apr 8, 2015 · Immunology

Device Facts

• Record ID: K150155
• Device Name: NOVA Lite DAPI ANA Kit
• Applicant: Inova Diagnostics, Inc.
• Product Code: DHN · Immunology
• Decision Date: Apr 8, 2015
• Decision: SESE
• Submission Type: Traditional
• Regulation: 21 CFR 866.5100
• Device Class: Class 2

Intended Use

NOVA Lite® DAPI ANA Kit is an indirect immunofluorescence assay for the qualitative detection and semi-quantitative determination of anti-nuclear antibodies of the IgG isotype in human serum by manual fluorescence microscopy or with the NOVA View Automated Fluorescence Microscope. 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. A trained operator must confirm results when generated with the NOVA View device.

Device Story

The NOVA Lite DAPI ANA Kit is an indirect immunofluorescence assay (IIFA) using HEp-2 cell substrate slides. Patient serum is incubated with HEp-2 cells; unbound antibodies are washed away. A FITC-conjugated anti-human IgG secondary antibody containing DAPI (a DNA-binding blue fluorescent dye) is applied. The DAPI dye enables automated imaging on the NOVA View system, while remaining invisible during traditional manual fluorescence microscopy. The device produces qualitative and semi-quantitative results (reactivity grades 0-4+). It is used in clinical laboratories by trained operators. The NOVA View system captures digital images of the slides, which the operator reviews on a computer monitor to confirm results. The output aids clinicians in diagnosing systemic rheumatic diseases by identifying the presence and pattern of autoantibodies.

Clinical Evidence

Clinical validation included 463 characterized samples (SLE, SSc, SS, MCTD, autoimmune myositis, healthy controls, etc.) tested at three sites. Primary endpoints were sensitivity, specificity, and agreement between manual, digital, and NOVA View interpretations. Sensitivity for SLE ranged 70.7–82.7% across sites/methods. Specificity (excluding healthy) ranged 67.2–77.0%. Overall agreement between NOVA View and manual reading was 87.0–89.8%. Pattern agreement between digital and manual reading was >90%.

Technological Characteristics

Materials: HEp-2 substrate slides, FITC-conjugated anti-human IgG, DAPI counterstain. Sensing: IIF microscopy. Energy: Mercury vapor or LED light source (460-495 nm excitation, 510-520 nm barrier). Connectivity: NOVA View system (networked/standalone). Software: NOVA View Software Version 2.1.3. Sterilization: Not applicable (reagents).

Indications for Use

Indicated for qualitative detection and semi-quantitative determination of IgG anti-nuclear antibodies (ANA) in human serum to aid in the diagnosis of systemic lupus erythematosus (SLE) and other systemic rheumatic diseases. For use by trained operators via manual fluorescence microscopy or the NOVA View Automated Fluorescence Microscope.

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

• NOVA Lite® HEp-2 ANA kit (k880736)

Submission Summary (Full Text)

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Image /page/0/Picture/1 description: The image shows the seal of the Department of Health & Human Services - USA. The seal features a stylized eagle with its wings spread, facing right. The eagle is composed of three curved lines that suggest feathers. The text "DEPARTMENT OF HEALTH & HUMAN SERVICES - USA" is arranged in a circular pattern around the eagle.

Food and Drug Administration 10903 New Hampshire Avenue Document Control Center - WO66-G609 Silver Spring, MD 20993-0002

April 8, 2015

INOVA DIAGNOSTICS, INC. c/o GABRIELLA LAKOS MD, PhD MEDICAL DIRECTOR, DIRECTOR OF ASSAY DEVELOPEMENT 9900 OLD GROVE ROAD SAN DIEGO, CA 92131

Re: k150155

Trade/Device Name: NOVA Lite® DAPI ANA Kit Regulation Number: 21 CFR 866.5100 Regulation Name: Antinuclear antibody immunological test system Regulatory Class: II Product Code: DHN, PIV Dated: January 22, 2015 Received: January 23, 2015

Dear Dr. Lakos:

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. The general controls provisions of the Act include requirements for annual registration, listing of devices, good manufacturing practice, labeling, and prohibitions against misbranding and adulteration. Please note: CDRH does not evaluate information related to contract liability warranties. We remind you, however, that device labeling must be truthful and not misleading.

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

Please be advised that FDA's issuance of a substantial equivalence determination does not mean that FDA has made a determination that your device complies with other requirements of the Act or any Federal statutes and regulations administered by other Federal agencies. You must comply with all the Act's requirements, including, but not limited to: registration and listing (21 CFR Part 807); labeling (21 CFR Parts 801 and 809); medical device reporting (reporting of medical device-related adverse events) (21 CFR 803); good manufacturing practice requirements as set forth in the quality systems (QS) regulation (21 CFR Part 820); and if applicable, the

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electronic product radiation control provisions (Sections 531-542 of the Act); 21 CFR 1000-1050.

If you desire specific advice for your device on our labeling regulations (21 CFR Parts 801 and 809), please contact the Division of Industry and Consumer Education at its toll-free number [phone] or [phone] or at its Internet address

http://www.fda.gov/MedicalDevices/Resourcesfor You/Industry/default.htm. 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

http://www.fda.gov/MedicalDevices/Safety/ReportaProblem/default.htm for the CDRH's Office of Surveillance and Biometrics/Division of Postmarket Surveillance.

You may obtain other general information on your responsibilities under the Act from the Division of Industry and Consumer Education at its toll-free number [phone] or [phone] or at its Internet address

http://www.fda.gov/MedicalDevices/ResourcesforYou/Industry/default.htm.

Sincerely yours.

# Leonthena R. Carrington -A

Leonthena R. Carrington, MS. MBA, MT(ASCP) Director Division of Immunology and Hematology Devices Office of In Vitro Diagnostics and Radiological Health Center for Devices and Radiological Health

Enclosure

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# Indications for Use

510(k) Number (if known) K150155

Device Name NOVA Lite® DAPI ANA Kit

#### Indications for Use (Describe)

NOVA Lite® DAPI ANA Kit is an indirect immunofluorescence assay for the qualitative detection and semi-quantitative determination of anti-nuclear antibodies of the IgG isotype in human serum by manual fluorescence microscopy or with the NOVA View Automated Fluorescence Microscope. 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. A trained operator must confirm results when generated with the NOVA View device.

Type of Use (Select one or both, as applicable)

| <span>☑</span> Prescription Use (Part 21 CFR 801 Subpart D) |
|-------------------------------------------------------------|
| <span>☐</span> Over-The-Counter Use (21 CFR 801 Subpart C) |

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Inova Diagnostics

# 510(k) Summary

# NOVA Lite® DAPI ANA Kit

### 510(k) Summary

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

### Table of Contents

| 1. | Submitter | 3 |
|-------|-------------------------------------------------------|----|
| 2. | Purpose of submission | 3 |
| 3. | Devices in the submission | 3 |
| 4. | Primary contact | 3 |
| 5. | Secondary contact | 3 |
| 6. | Product Information and Classification | 3 |
| 6.1 | Proprietary and established names | 3 |
| 6.2 | Regulatory information. | 4 |
| 6.3 | Regulatory information. | 4 |
| 7. | Intended Use | 4 |
| 8. | Indications for Use | 5 |
| 9. | Predicate device | 5 |
| 10. | Comparison matrix to predicate device. | 5 |
| 11. | Type of the product | 5 |
| 12. | Device description. | 6 |
| 13. | Principle of the method and summary of the procedure. | 6 |
| 14. | Analytical performance characteristics. | 6 |
| 15.1 | NOVA View system | 6 |
| 15.2. | Nomenclature used in the studies | 7 |
| 15.3 | Limit of Blank (LoB), Limit of Detection (LoD). | 7 |
| 15.4 | Precision performance. | 7 |
| | Repeatability and reproducibility. | 8 |
| 15.4 | Linearity and Analytical Measuring Range (AMR) | 10 |

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| 15.5 | Interference | 10 |
|------|-----------------------------------------------------------------------------------------|----|
| 15.6 | Cross-reactivity | 10 |
| 15.7 | Lot to lot comparison | 11 |
| 15.8 | Accelerated stability study of the anti-human IgG-FITC conjugate with DAPI (P/N 508102) | 13 |
| 16. | Cutoff | 14 |
| 17. | Comparison with the predicate device | 14 |
| 17.1 | Conjugate comparison | 14 |
| 17.2 | Method comparison | 15 |
| 18. | Agreement between digital reading results and manual reading | 17 |
| 18.1 | Accuracy of endpoint titration | 17 |
| 18.2 | Agreement on clinical sample cohort | 18 |
| 19. | Clinical performance | 19 |
| 19.1 | Clinical sensitivity and specificity | 19 |
| 20. | Expected values | 25 |
| 21. | CDC ANA reference sera | 26 |

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#### 1. Submitter

Inova Diagnostics, Inc 9900 Old Grove Road, San Diego, CA, 92131

#### 2. Purpose of submission

New device

#### 3. Devices in the submission

NOVA Lite® DAPI ANA Kit

#### 4. Primary contact

Gabriella Lakos, Director of Research and Development Inova Diagnostics, Inc 9900 Old Grove Road, San Diego, CA, 92131 Phone: [phone] email: [e-mail]

#### 5. Secondary contact

Ronda Elliott, VP of Quality Systems and Regulatory Affairs Inova Diagnostics, Inc 9900 Old Grove Road, San Diego, CA, 92131 Phone: [phone] Fax: [phone] email: [e-mail]

#### 6. Product Information and Classification

#### 6.1 Proprietary and established names

Proprietary name: NOVA Lite® DAPI ANA Kit

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| Common name: | Antinuclear antibody kit |
|----------------------|--------------------------------------------------------------------|
| Classification name: | antinuclear antibody, indirect immunofluorescent, antigen, control |

#### 6.2 Regulatory information

| Regulation Description | Antinuclear antibody immunological test system |
|------------------------------|----------------------------------------------------------------------------------|
| Regulation Medical Specialty | Immunology |
| Review Panel | Immunology |
| Product Code | DHN |
| Regulation Number | 866.5100 |
| Device Class | II |
| 6.3 Regulatory information | |
| Regulation Description | Automated indirect immunofluorescence<br>microscope and software-assisted system |
| Regulation Medical Specialty | Immunology |
| Review Panel | Immunology |
| Product Code | PIV |
| Regulation Number | 866.4750 |

ll

#### 7. Intended Use

Device Class

NOVA Lite® DAPI ANA Kit is an indirect immunofluorescence assay for the qualitative detection and semi-quantitative determination of anti-nuclear antibodies of the IgG isotype in human serum by manual fluorescence microscopy or with the NOVA View Automated Fluorescence Microscope. 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. A trained operator must confirm results when generated with the NOVA View device.

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### 8. Indications for Use

Same as intended use.

### 9. Predicate device

NOVA Lite HEp-2 ANA Kit, 510k number: k880736

#### 10. Comparison matrix to predicate device

| Item | NOVA Lite DAPI ANA Kit | Predicate Device |
|-----------------------------|--------------------------------------------------------------------------------------------------------------------|-----------------------------------------------------------------------------------------------|
| Intended use | qualitative detection and semi-quantitative determination of anti-nuclear antibodies of IgG isotype in human serum | screening and semi-quantitative determination of anti-nuclear antibodies (ANA) in human serum |
| Analyte | Anti-nuclear antibodies of IgG isotype | Anti-nuclear antibodies of IgG isotype |
| Assay methodology | indirect immunofluorescence assay | indirect immunofluorescence assay |
| Interpretation | by manual fluorescence microscopy or with the NOVA View device | by manual fluorescence microscopy |
| Antigen | HEp-2 cells substrate, 12-well slides | HEp-2 cells substrate, 12-well slides |
| Sample type | Serum | Serum |
| Sample dilution | 1:80 | 1:40 |
| Conjugate | FITC conjugated anti-human IgG (Fc specific) with added 4',6-diamidino-2-phenylindole (DAPI) | FITC conjugated anti-human IgG (Fc specific) |
| Additional dye in Conjugate | 4',6-diamidino-2-phenylindole (DAPI) | None |
| Controls | Positive (ANA Titratable) and Negative Control | Positive (ANA Titratable) and Negative Control |
| Storage | 2-8 °C | 2-8 °C |
| Shelf life | 12 months | 24 months |

### 11. Type of the product

Assay/reagents including controls.

Qualitative and semi-quantitative.

Device technology: indirect immunofluorescence.

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### 12. Device description

The NOVA Lite DAPI ANA Kit is an indirect immunofluorescence assay for the detection and semiquantitative determination of anti-nuclear antibodies in human serum.

Kit components:

- HEp-2 (human epithelial cell) substrate slides; 12 wells/slide, with desiccant.
- FITC IgG Conjugate with DAPI, containing 0.09% sodium azide; ready to use.
- Positive Control: ANA Titratable Pattern, human serum with antibodies to HEp-2 nuclei in buffer, containing 0.09% sodium azide; pre-diluted, ready to use.
- . Negative Control: IFA System Negative Control, diluted human serum with no ANA present, containing 0.09% sodium azide; pre-diluted, ready to use.
- PBS II (40x) Concentrate, sufficient for making 2000 mL of 1x PBS II.
- Mounting Medium, containing 0.09% sodium azide ●
- Coverslips

### 13. Principle of the method and summary of the procedure

Samples are diluted 1:80 in PBS and incubated with the antigen substrate (HEp-2 cells). After incubation, unbound antibodies are washed off. The substrate is then incubated with anti-human IgG-FITC conjugate. The conjugate contains a DNA-binding blue fluorescent dye, 4',6-diamidino-2-phenylindole (DAPI) that is required for NOVA View use. The blue dye is not visible by traditional flurescence microscope at the wavelength where FITC fluorescence is viewed. Unbound reagent is washed off, and the slides are coverslipped. Stained slides are read by manual fluorescence microscopy, or are scanned with the NOVA View Automated Fluorescence Microscope. The resulting digital images are reviewed and interpreted from the computer monitor. ANA positive samples will exhibit an apple green fluorescence corresponding to areas of the cell nuclei where autoantibody has bound. Some sera may contain antibodies reacting with cytoplasmic antigens, and will exhibit apple green fluorescence corresponding to areas of the cell cytoplasm where autoantibody has bound. When slides are assessed by NOVA View, digital images of representative areas of the well are captured. These digital images must be reviewed and interpreted from the computer monitor by a trained operator. Samples that are positive at 1:80 may be titered by performing a 2-fold serial dilution from the initial screening dilution with PBS buffer (i.e. 1:80, 1:160, 1:640, 1:1280, 1:2560, etc.) to determine the endpoint titer.

### 14. Analytical performance characteristics

#### 15.1 NOVA View Device

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The performance characteristics of the NOVA View device are included in a separate submission, DEN140039.

#### 15.2. Nomenclature used in the studies

All studies have been performed by interpreting the results with both manual microscopy and with the NOVA View system.

"Manual" and "Manual reading" refers to results obtained by reading the slides with traditional fluorescence microscope.

"NOVA View" refers to raw results obtained with the NOVA View Automated Fluorescence Microscope, such as Light Intensity Units (LIU), positive/negative classification and pattern information.

"Digital", "Digital reading" and "Digital image" refers to results obtained by reading NOVA View generated images on the computer monitor.

If only cytoplasmic pattern is present, the ANA is considered negative.

For statistical calculations, a positive result is presented as "1", and a negative result is presented as "0".

Intensity of the staining is expressed in reactivity grades. Grade 0 is negative; grades 1-4 are weak to strong positive.

When an operator had to read the same set of slides twice (for example, with manual microscope and on the computer monitor), a minimum 3-day "washout" period was included between the readings.

### ANA pattern nomenclature:

| Patterns reported by NOVA View | |
|--------------------------------|--------------|
| H | Homogeneous |
| S | Speckled |
| N | Nucleolar |
| C | Centromere |
| D | Dots |
| U | Unrecognized |

Limit of Blank (LoB), Limit of Detection (LoD) 15.3

N/A

15.4 Precision performance

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#### Repeatability and reproducibility

To assess the precision performance of the NOVA Lite DAPI ANA Kit results, a study was performed by processing 3 negative and 10 positive samples ("first set") with various patterns and intensities with NOVA Lite DAPI ANA kit, in three replicates, in 10 runs (2 runs per day), resulting in 30 data points for each sample. The slides were read with NOVA View, and digital images were interpreted by the operator. Slides in this study were not read with manual microscope; i.e. two set of results were generated: NOVA View output and digital image reading results.

Additional precision study ("second set") was performed on 22 samples: 20 negative and around-the cut-off samples, and 2 samples with 3+ average grade intensity level. Samples were tested in three replicates, in 10 runs (2 runs per day), resulting in 30 data points for each sample. The slides were read with NOVA View, and digital images were interpreted, moreover, slides were read with manual microscope, too; i.e. three set of results were generated: NOVA View output, digital image reading results and manual reading results.

A third, separate study has previously been performed ("third set") with samples tested in triplicates or duplicates, in 5 runs, resulting in 15 or 10 data points for each sample. The slides were read with NOVA View, and digital images were interpreted, moreover, slides were read with manual microscope, too; i.e. three set of results were generated: NOVA View output, digital image reading results and manual reading results.

Acceptance criteria: Difference between reactivity grades within one run (between replicates) are within ± one reactivity grade. Average reactivity grade difference between any runs is within ± one reactivity grade.

Results: For both digital image reading and manual reading, grades were within ± one reactivity grade within one run (within triplicates), and the average grade was no more than one reactivity grade different between runs. Pattern was consistent for 100% of the replicates (considering positive results only).

The results of the above three precision studies are summarized in the Table below. Samples in the Table are sorted according to NOVA View LIU values.

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# NOVA Lite® DAPI ANA Kit

| | Sample ID | n | Mean LIU | NOVA View output | | Manual reading | | | Digital reading | | |
|------------|-------------------------|-------|----------|------------------|------------|------------------------|------------|------------|------------------------|------------|------------|
| | | | | % negative | % positive | Reactivity grade range | % negative | % positive | Reactivity grade range | % negative | % positive |
| first set | NVB012 | 30 | 4.7 | 100 | 0 | N/T | N/T | N/T | 0 | 100 | 0 |
| | NVB007 | 30 | 7.6 | 100 | 0 | N/T | N/T | N/T | 0 | 100 | 0 |
| | NVB063 | 30 | 7.9 | 100 | 0 | N/T | N/T | N/T | 0 | 100 | 0 |
| | NVB111 | 30 | 38.5 | 63.3 | 36.7 | N/T | N/T | N/T | 0-1 | 3.3 | 96.7 |
| | NVB079 | 30 | 91.6 | 13.3 | 86.7 | N/T | N/T | N/T | 0-1 | 3.3 | 96.7 |
| | NVB009 | 30 | 229.1 | 0 | 100 | N/T | N/T | N/T | 2-3 | 0 | 100 |
| | NVB029 | 30 | 233.8 | 0 | 100 | N/T | N/T | N/T | 4 | 0 | 100 |
| | NVB087 | 30 | 310.5 | 0 | 100 | N/T | N/T | N/T | 1-2 | 0 | 100 |
| | NVB017 | 30 | 310.6 | 0 | 100 | N/T | N/T | N/T | 1-2 | 0 | 100 |
| | NVB023 | 30 | 715.5 | 0 | 100 | N/T | N/T | N/T | 4 | 0 | 100 |
| | NVB004 | 30 | 933.3 | 0 | 100 | N/T | N/T | N/T | 4 | 0 | 100 |
| | NVB118 | 30 | 1300.1 | 0 | 100 | N/T | N/T | N/T | 4 | 0 | 100 |
| | NVB037 | 30 | 2217.7 | 0 | 100 | N/T | N/T | N/T | 4 | 0 | 100 |
| second set | NV20 | 30 | 3.5 | 100 | 0 | 0 | 100 | 0 | 0 | 100 | 0 |
| | NV16 | 30 | 10.2 | 100 | 0 | 0 | 100 | 0 | 0 | 100 | 0 |
| | NV2 | 30 | 11.4 | 100 | 0 | 0 | 100 | 0 | 0 | 100 | 0 |
| | NV8 | 30 | 13.5 | 100 | 0 | 0 | 100 | 0 | 0 | 100 | 0 |
| | NV15 | 30 | 16.6 | 100 | 0 | 0-1 | 13.3 | 86.7 | 0-1 | 16.7 | 83.3 |
| | NV9 | 30 | 19.1 | 100 | 0 | 0-1 | 46.7 | 53.3 | 0 | 100 | 0 |
| | SB24216 | 30 | 31.4 | 86.7 | 13.3 | 1 | 0 | 100 | 1 | 0 | 100 |
| | NV22 | 30 | 33.6 | 76.7 | 23.3 | 0-1 | 96.7 | 3.3 | 0-1 | 76.7 | 23.3 |
| | NV26 | 30 | 38.4 | 90.0 | 10.0 | 0-1 | 60.0 | 40.0 | 0-1 | 53.3 | 46.7 |
| | NV14 | 30 | 38.8 | 43.3 | 56.7 | 0-1 | 6.7 | 93.3 | 1 | 0 | 100 |
| | NV13 | 30 | 40.5 | 66.7 | 33.3 | 1 | 0 | 100 | 0-1 | 6.7 | 93.3 |
| | NV5 | 30 | 40.7 | 66.7 | 33.3 | 1-2 | 0 | 100 | 0-1 | 16.7 | 83.3 |
| NVB440 | 30 | 43.8 | 73.3 | 26.7 | 0-1 | 33.3 | 66.7 | 0-1 | 46.7 | 53.3 | |
| NV4 | 30 | 57.5 | 43.3 | 56.7 | 1-2 | 0 | 100 | 1-2 | 0 | 100 | |
| NVB201 | 30 | 62.8 | 26.7 | 73.3 | 1-2 | 0 | 100 | 1-2 | 0 | 100 | |
| NVB074 | 30 | 63.8 | 16.7 | 83.3 | 1 | 0 | 100 | 1 | 0 | 100 | |
| NV12 | 30 | 64.8 | 36.7 | 63.3 | 1-2 | 0 | 100 | 1-2 | 0 | 100 | |
| NVB369 | 30 | 72.4 | 23.3 | 76.7 | 0-1 | 3.3 | 96.7 | 0-1 | 13.3 | 86.7 | |
| NV7 | 30 | 74.1 | 10.0 | 90.0 | 1-2 | 0 | 100 | 1-2 | 0 | 100 | |
| NV10 | 30 | 128.5 | 30.0 | 70.0 | 1-2 | 0 | 100 | 1-2 | 0 | 100 | |
| NV23 | 30 | 822.4 | 0 | 100 | 2-3 | 0 | 100 | 3 | 0 | 100 | |
| NV6 | 30 | 903.9 | 0 | 100 | 3 | 0 | 100 | 3 | 0 | 100 | |
| third set | PMDx 5087 | 15 | 24.6 | 100 | 0 | 100 | 0 | 100 | 0 | 100 | 0 |
| | SS-A Monospecific 08203 | 15 | 103.6 | 0 | 100 | 2 | 0 | 100 | 1-2 | 0 | 100 |
| | AMA 930328 | 15 | 882.6 | 0 | 100 | 4 | 0 | 100 | 4 | 0 | 100 |
| | Centromere 120571 | 10 | 1052.9 | 0 | 100 | 3-4 | 0 | 100 | 4 | 0 | 100 |
| | Nucleolar 120559 | 10 | 1339.8 | 0 | 100 | 3 | 0 | 100 | 4 | 0 | 100 |
| | DNA PS0007 520847 | 15 | 1375.6 | 0 | 100 | 3-4 | 0 | 100 | 4 | 0 | 100 |
| | ANA DNA 420530 | 10 | 1607.8 | 0 | 100 | 4 | 0 | 100 | 4 | 0 | 100 |
| | SmRNP 220951 | 10 | 2811.2 | 0 | 100 | 3 | 0 | 100 | 4 | 0 | 100 |

N/T: Not tested

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#### 15.4 Linearity and Analytical Measuring Range (AMR)

N/A

#### 15.5 Interference

The interference study was performed according to CLSI EPO7-A2, Interference Testing in Clinical Chemistry; Approved Guideline - Second Edition.

Interference by bilirubin, hemoglobin, triglycerides, cholesterol and RF IgM was assessed using the following materials and concentrations. The Table below contains all three concentration levels that were tested:

| Interfering substance | Manufacturer Part<br>number | Lot number | Concentration #1<br>tested | Concentration #2<br>tested | Concentration #3<br>tested |
|-----------------------|-----------------------------|------------|----------------------------|----------------------------|----------------------------|
| Bilirubin, conjugated | EMD 201102 | DU3038000 | 10 mg/dL | 5 mg/dL | 2.5 mg/dL |
| Hemoglobin | SIGMA H7379 | 051M7004V | 200 mg/dL | 100 mg/dL | 50 mg/dL |
| Triglicerydes | SCIPAC P235-8 | 1201-146 | 1000 mg/dL | 500 mg/dL | 250 mg/dL |
| Cholesterol | SCIPAC P235-8 | 1201-146 | 224.3 mg/dL | 112.1 mg/mL | 56 mg/mL |
| RF IgM | QC #16 | ZG0024783 | 56 AU | 33.6 AU | 11.2 AU |

Three specimens were tested (one negative, one medium positive, and one strong positive). Interfering substances (hemoglobin, bilirubin, triglycerides, cholesterol) were spiked into every specimen at three different concentrations (see above) in 10% of total specimen volume, and the resulting samples were assessed in triplicates according to the standard protocol (diluted in 1:80 and processed on HEp-2 slides). The concentrations shown above are final (testing) concentrations in the sample after spiking. To assess interference with rheumatoid factor (RF), 10%, 30% and 50% (volume) RF positive sample was added to the test samples were processed with NOVA Lite DAPI ANA kit, and read with NOVA View. Digital images were interpreted and confirmed. Moreover, all slides were read by the same operator with manual microscopy. Appropriate controls were made by adding 10% (volume) sample diluent to the same samples and for testing for RF interference, adding 10%, 30% and 50% (volume) sample diluent to the samples.

Reactivity grades of samples containing the interfering substance were within ± one grade of the control samples with both manual and digital reading.

No interference was detected with bilirubin up to 10 mg/dL, hemoglobin up to 200 mg/dL, triglycerides up to 1000 mg/dL, cholesterol up to 224.3 mg/dL and RF IgM up to 56 AU.

#### 15.6 Cross-reactivity

Cross reactivity was examined in the following patients with autoimmune thyroid disease/TPO antibodies, patients with celiac disease/anti-tTG antibodies, patients with anti-MPO and anti-PR3 antibodies, patients with Crohn's/inflammatory bowel disease, and patients with rheumatoid arthritis.

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The number and distribution of the population is shown in the Table below, together with the ANA positivity rate. Considering all 114 samples, the observed positivity rate was 25% for NOVA View results, 27% for digital image reading, and 28% for manual reading. The positivity rate was 15% for NOVA View results, 19% for digital image reading, and 21% for manual reading, when rheumatoid arthritis samples were not included. This positivity rate is in line with the expected results and the published literature. ANA positivity in RA has previously been described with high frequency. RA-33 antibodies are present in up to 36% of RA patients, and anti-histone antibodies have also been identified in the sera of RA patients.

| Cross-reactivity cohort, n=114 | | Positivity rate | | | | | |
|---------------------------------------|--------|-----------------|-----|----------------|-----|-----------------|-----|
| Sample type | Number | NOVA View | | Manual reading | | Digital reading | |
| | | Number | % | Number | % | Number | % |
| Anti-MPO/anti-PR3 | 26 | 1 | 4% | 3 | 12% | 4 | 15% |
| Crohn's/Inflammatory<br>bowel disease | 20 | 8 | 40% | 9 | 45% | 8 | 40% |
| Autoimmune<br>thyroiditis | 24 | 2 | 8% | 4 | 17% | 3 | 13% |
| Celiac disease | 24 | 3 | 13% | 4 | 17% | 3 | 13% |
| Rheumatoid arthritis | 20 | 15 | 75% | 11 | 55% | 14 | 70% |
| Total | 114 | 29 | 25% | 31 | 28% | 32 | 27% |

#### 15.7 Lot to lot comparison

Lot to lot comparison study was performed on three reagent lots. Forty sera were tested along with altogether 8 controls (one Negative and one Positive Control on each slide).

The following comparisons were made:

- . NOVA view output: negative/positive, LIU and pattern comparison.
- . Digital image reading: negative/positive, grade and pattern comparison
- Manual image reading: negative/positive, grade and pattern comparison.

### Agreement:

Average positive, average negative and total agreement between digital image reading result and manual reading results between the three lots were calculated, and the results are shown below:

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

## NOVA Lite® DAPI ANA Kit

| Digital reading | Lot 008559 vs Lot<br>009398 | Lot 008559 vs Lot<br>009399 | Lot 009398 vs Lot<br>009399 |
|----------------------------|-----------------------------|-----------------------------|-----------------------------|
| Average negative agreement | 94.7 (85.7-100.0) | 91.9 (80.9-100.0) | 97.4 (90.9-100.0) |
| Average positive agreement | 95.2 (87.2-100.0) | 93.0 (83.3-100.0) | 97.6 (91.4-100.0) |
| Total agreement | 95.0 (83.1-99.4) | 92.5 (79.6-94.4) | 97.5 (86.8-99.9) |

| Manual reading | Lot 008559 vs Lot<br>009398 | Lot 008559 vs Lot<br>009399 | Lot 009398 vs Lot<br>009399 |
|----------------------------|-----------------------------|-----------------------------|-----------------------------|
| Average negative agreement | 93.8 (82.8-100.0) | 93.8 (82.8-100.0) | 100 (100-100) |
| Average positive agreement | 95.8 (88.9-100.0) | 95.8 (88.9-100.0) | 100 (100-100) |
| Total agreement | 95.0 (83.1-99.4) | 95.0 (83.1-99.4) | 100 (91.2-100) |

### Grade agreement:

All grades (100%) were within ± 1 grade from each other for all samples in any pair-wise comparisons with manual and digital reading:

| Digital reading grade agreement | | | | | | | Manual reading grade agreement | | | | | | | |
|---------------------------------|------------|---|---|------------|---|-------|--------------------------------|----|------------|---|------------|---|-------|--|
| | | | | | | | | | | | | | | |
| | Lot 008559 | | | | | | Lot 008559 | | | | | | | |
| Lot 009398 | 0 | 1 | 2 | 3 | 4 | Total | Lot 009398 | 0 | 1 | 2 | 3 | 4 | Total | |
| 0 | 18 | 2 | 0 | 0 | 0 | 20 | 0 | 15 | 0 | 0 | 0 | 0 | 15 | |
| 1 | 0 | 3 | 0 | 0 | 0 | 3 | 1 | 2 | 5 | 0 | 0 | 0 | 7 | |
| 2 | 0 | 1 | 2 | 0 | 0 | 3 | 2 | 0 | 1 | 5 | 0 | 0 | 6 | |
| 3 | 0 | 0 | 1 | 8 | 0 | 9 | 3 | 0 | 0 | 0 | 6 | 1 | 7 | |
| 4 | 0 | 0 | 0 | 0 | 5 | 5 | 4 | 0 | 0 | 0 | 1 | 4 | 5 | |
| Total | 18 | 6 | 3 | 8 | 5 | 40 | Total | 17 | 6 | 5 | 7 | 5 | 40 | |
| | | | | | | | | | | | | | | |
| | | | | | | | | | | | | | | |
| | | | | Lot 008559 | | | | | | | Lot 008559 | | | |
| Lot 009399 | 0 | 1 | 2 | 3 | 4 | Total | Lot 009399 | 0 | 1 | 2 | 3 | 4 | Total | |
| 0 | 17 | 2 | 0 | 0 | 0 | 19 | 0 | 15 | 0 | 0 | 0 | 0 | 15 | |
| 1 | 1 | 4 | 0 | 0 | 0 | 5 | 1 | 2 | 6 | 0 | 0 | 0 | 8 | |
| 2 | 0 | 0 | 2 | 0 | 0 | 2 | 2 | 0 | 0 | 5 | 1 | 0 | 6 | |
| 3 | 0 | 0 | 1 | 8 | 0 | 9 | 3 | 0 | 0 | 0 | 5 | 0 | 5 | |
| 4 | 0 | 0 | 0 | 0 | 5 | 5 | 4 | 0 | 0 | 0 | 1 | 5 | 6 | |
| Total | 18 | 6 | 3 | 8 | 5 | 40 | Total | 17 | 6 | 5 | 7 | 5 | 40 | |
| | | | | | | | | | | | | | | |
| | | | | | | | | | | | | | | |
| | | | | Lot 009398 | | | | | Lot 009398 | | | | | |
| Lot 009399 | 0 | 1 | 2 | 3 | 4 | Total | Lot 009399 | 0 | 1 | 2 | 3 | 4 | Total | |
| 0 | 19 | 0 | 0 | 0 | 0 | 19 | 0 | 15 | 0 | 0 | 0 | 0 | 15 | |
| 1 | 1 | 3 | 1 | 0 | 0 | 5 | 1 | 0 | 7 | 1 | 0 | 0 | 8 | |
| 2 | 0 | 0 | 2 | 0 | 0 | 2 | 2 | 0 | 0 | 5 | 1 | 0 | 6 | |
| 3 | 0 | 0 | 0 | 9 | 0 | 9 | 3 | 0 | 0 | 0 | 5 | 0 | 5 | |
| 4 | 0 | 0 | 0 | 0 | 5 | 5 | 4 | 0 | 0 | 0 | 1 | 5 | 6 | |
| Total | 20 | 3 | 3 | 9 | 5 | 40 | Total | 15 | 7 | 6 | 7 | 5 | 40 | |

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

### Pattern agreement:

Pattern agreement was assessed in pair-wise comparison between lots. Only definitive patterns (Homogeneous, Speckled, Centromere, Nuclear dots) were considered as pattern agreement. User reported "Other" patterns were not considered as an agreement.

There was 100% pattern agreement between the lots with manual and digital image reading, considering positive samples only.

#### 15.8 Accelerated stability study of the anti-human IgG-FITC conjugate with DAPI (P/N 508102)

Accelerated stability study was performed on three lots of conjugate with DAPI according to an isochronous design, at 37 ± 3°C for 4 weeks. Each week a new vial of sealed component was placed in the incubator at 37 ± 3°C, and all components were tested at the end of the study period together with a vial that was stored at 5 ± 3℃ (control). Testing was performed by staining the slides with characterized samples. All calculations were performed by comparing results obtained with the control vial (stored at 5 ± 3℃) to those obtained with vials stored at 37 ± 3℃ for 1, 2, 3, and 4 weeks, where one week is equal to six months at 5 ± 3°C. All slides were read with NOVA View; moreover, all slides were read by the same operator with manual microscopy.

Acceptance criteria for one year preliminary shelf life:

Reactivity grades obtained on slides stored at 37 °C for 2 weeks are within ± one grade of those obtained on the control slides.

| | L/N: 042194P1 | | L/N: 042195P2 | | L/N: 042196V3 | |
|------------------------------|---------------|---------------|---------------|---------------|---------------|---------------|
| | Control | Week2 | Control | Week2 | Control | Week2 |
| Sample ID | grade pattern | grade pattern | grade pattern | grade pattern | grade pattern | grade pattern |
| Homogeneous Positive Control | 3 H | 4 H | 3 H | 3 H | 3 H | 3 H |
| IFA System Negative Control | 0 n/a | 0 n/a | 0 n/a | 0 n/a | 0 n/a | 0 n/a |
| Speckled Positive Control | 3 S | 3 S | 3 S | 4 S | 4 S | 4 S |
| Nucleolar Positive Control | 3 N | 3 N | 3 N | 3 N | 3 N | 3 N |
| Centromere Positive Control | 3 C | 3 C | 3 C | 3 C | 3 C | 3 C |
| AMA Positive Control | 4 D | 3 D | 3 D | 4 D | 4 D | 3 D |
| PS0002 420530 (Homogeneous) | 1 H | 1 H | 1 H | 1 H | 1 H | 1 H |
| PS0005 010047 (Negative) | 0 n/a | 0 n/a | 0 n/a | 0 n/a | 0 n/a | 0 n/a |
| PS0008 220951 (Speckled) | 1 S | 0 | 1 S | 1 S | 1 S | 1 S |
| PS0006 120559 (Nucleolar) | 1 N | 1 N | 2 N | 2 N | 2 N | 2 N |
| PS0004 120571 (Centromere | 1 C | 1 C | 1 C | 1 C | 1 C | 1 C |
| PS0003 930328 (AMA) | 2 D | 1 D | 1 D | 1 D | 2 D | 1 D |

Results are summarized in the table below. Acceptance criteria were met.

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

#### 16. Cutoff

The serum dilution of 1:80 was selected to provide optimal clinical sensitivity and specificity. The performance of this serum dilution has been validated as described in sections "Clinical performance" and "Expected values".

#### 17. Comparison with the predicate device

#### 17.1 Conjugate comparison

The NOVA Lite DAPI ANA Kit contains the same components as the predicate device, with the exception of the conjugate. To adapt the assay for use on NOVA View, the blue fluorescent dye DAPI (4',6diamidino-2-phenylindole) that binds strongly to A-T rich regions in DNA was added to the conjugate. The addition of DAPI does not influence the test utility and performance when used manually, as it is not visible at the wavelength used for reading slides with traditional fluorescence microscope, and does not interfere with antibody binding.

To demonstrate the equivalent performance of the conjugate with and without DAPI, a comparison study has been performed on clinical samples.

407 individual serum samples have been tested. Two sets of slides were stained: one with the conjugate without DAPI (P/N: 508113, included in the predicate device) the other with the conjugate with DAPI (508102). The 1:80 serum dilution was used. The two sets of slides were read by the same operator with an Olympus CX31 fluorescent microscope. Positive/negative agreement and grade correlation were evaluated.

### Acceptance criteria:

Agreement between the two sets is > 85%.

Pattern agreement (for positive samples only) is > 85%.

Grades are within ± one grade from each other for 90% of the samples.

All acceptance criteria were met. Total agreement between the two sets was 96.6%. All grades were within ± one grade from each other. 210 samples were positive with both sets; pattern discrepancy was observed in three cases in those samples that were interpreted as positive with both conjugates.

| (N=407) | 508113 (conjugate w/o DAPI) | | | | Percent Agreement<br>(95% Confidence) |
|-----------------------------------|-----------------------------|----------|-------|-----|---------------------------------------|
| | Negative | Positive | Total | | |
| 50102<br>(conjugate<br>with DAPI) | Negative | 183 | 3 | 186 | Pos. Agreement: 98.6% (95.9-99.7%) |
| | Positive | 11 | 210 | 221 | Neg. Agreement: 94.3% (90.1-97.1%) |
| | Total | 194 | 213 | 407 | Total Agreement: 96.6% (94.3-98.1%) |

### Agreement:

Grade agreement:

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

| | Grades, 508113 (conjugate wo DAPI) | | | | | |
|----------------------------------------|------------------------------------|----|----|----|----|-------|
| Grades, 50102<br>(conjugate with DAPI) | 0 | 1+ | 2+ | 3+ | 4+ | Total |
| 0 | 183 | 3 | 0 | 0 | 0 | 186 |
| 1+ | 11 | 71 | 8 | 0 | 0 | 90 |
| 2+ | 0 | 14 | 73 | 3 | 0 | 90 |
| 3+ | 0 | 0 | 2 | 26 | 2 | 30 |
| 4+ | 0 | 0 | 0 | 2 | 9 | 11 |
| Total | 194 | 88 | 83 | 31 | 11 | 407 |

#### 17.2 Method comparison

Results that were obtained with the NOVA Lite DAPI ANA kit, using 1:80 serum dilution, were compared to those obtained with the reference device (1:40 serum dilution, and anti-human IgG conjugate without DAPI).

The comparison study was performed on 410 samples: 400 clinically characterized sera, and 10 samples with known ANA patterns. All slides were interpreted with traditional fluorescence microscopy only. Interpretation included positive/negative categorization, pattern interpretation and grading of positive samples on a scale of 1+ to 4+.

The distribution of the cohort and the frequency of positive results are shown in the Table below:

| | Number of<br>samples | Number of<br>positive at 1:40 | Number of<br>positive at 1:80 |
|----------------------------------------|----------------------|-------------------------------|-------------------------------|
| Apparently healthy controls | 150 | 41 | 17 |
| SLE (Systemic Lupus Erythematosus) | 100 | 85 | 81 |
| SS (Sjogren's syndrome) | 30 | 23 | 21 |
| SSc (Systemic Sclerosis) | 30 | 20 | 15 |
| Idiopathic inflammatory myositis (IIM) | 10 | 9 | 7 |
| MCTD (Mixed Connective Tissue Disease) | 20 | 12 | 12 |
| Infectious disease | 30 | 6 | 4 |
| RA (Rheumatoid arthritis) | 30 | 20 | 17 |
| Centromere antibody positive | 5 | 5 | 5 |
| Mitochondrial antibody positive | 5 | 4 | 5 |
| Total | 410 | 224 | 184 |

{18}------------------------------------------------

Agreement between the two methods is shown below:

| | Predicate device, 1:40 | | |
|---------------------------------|------------------------|----------|-------|
| NOVA Lite DAPI ANA<br>Kit, 1:80 | Negative | Positive | Total |
| Negative | 181 | 45 | 226 |
| Positive | 5 | 179 | 184 |
| Total | 186 | 224 | 410 |

| | Positive Agreement %<br>(95% CI) | Negative Agreement %<br>(95% CI) | Total Agreement %<br>(95% CI) |
|-----------------------|----------------------------------|----------------------------------|-------------------------------|
| 1:80 vs 1:40 dilution | 79.9 (74.1-85.0) | 97.3 (91.5-100.0) | 87.7 (84.2-90.8) |

#### Pattern agreement

179 samples were positive according to both dilutions. The number of discrepant samples (not including negative/positive discrepancies, but including patterns interpreted as "other") was five (2.2% of samples that tested positive in 1:40 dilution).

### Grade agreement

Fluorescence intensity grades were within ± one grade from each other for 407 samples (99.5%). Grade agreement is shown in the matrix below:

| Fluorescence grade<br>NOVA Lite DAPI<br>ANA Kit | Fluorescence grade, predicate device | | | | | |
|-------------------------------------------------|--------------------------------------|----|----|----|----|-------|
| | 0 | 1+ | 2+ | 3+ | 4+ | Total |
| 0 | 181 | 44 | 1 | 0 | 0 | 226 |
| 1+ | 3 | 35 | 31 | 0 | 0 | 69 |
| 2+ | 1 | 3 | 37 | 24 | 0 | 65 |
| 3+ | 0 | 0 | 0 | 14 | 11 | 25 |
| 4+ | 0 | 0 | 0 | 1 | 23 | 24 |
| Total | 185 | 82 | 69 | 39 | 34 | 409* |

*Grade was not reported for one sample

Out of the 410 samples, 45 samples that were positive in 1:40 dilution were negative in 1:80. Out of the 45 samples, 28 were in from the healthy and infectious diseases population, and 3 had the diagnosis of RA. Fourteen samples were from patients with ANA-associated autoimmune diseases. Two samples

{19}------------------------------------------------

were from patients with IIM, two from patients with Sjogren's syndrome, five had systemic sclerosis. All these samples had a fluorescence intensity grade of 1+, except for one myositis sample that had a grade of 2+.

The prevalence of ANA in the healthy population (n=150) was 27.3% when sera were tested in 1:40 dilution, and was 11.3% when sera were tested in 1:80 dilution.

| | Sensitivity % (95% CI) | | Specificity* % |
|---------------|------------------------|------------------|------------------|
| | SLE | SARD | (95% CI) |
| | (N=100) | (N=190) | (N=60) |
| 1:40 dilution | 85.9 (77.4-92.0) | 78.8 (72.3-84.4) | 56.7 (43.2-69.4) |
| 1:80 dilution | 80.8 (71.7-88.0) | 71.4 (64.4-77.8) | 65.0 (51.6-76.9) |

Overall clinical sensitivity and specificity is shown below:

SARD: Systemic Autoimmune Rheumatic Disease (includes SLE, SSc, SS, MCTD and IIM)

*Control samples include RA and infectious disease population

#### 18. Agreement between digital reading results and manual reading

#### 18.1 Accuracy of endpoint titration

To assess the accuracy of the system at low analyte levels (around the cut-off), 10 ANA positive samples with various ANA intensities and IIF patterns were titered to endpoint with NOVA Lite DAPI ANA kit at Inova (Site #1) and at two external sites (shown as Site #3) as part of the validation study. The same samples were tested at each site. Samples were diluted in PBS starting at 1:80, and diluted 2fold until a dilution of 1:40,960 was reached (10 dilutions per sample). All samples were read with NOVA View. Digital images were interpreted and confirmed, and the endpoint titer (the dilution of the last positive result) was determined for each sample. Moreover, all slides were read by the same operator with manual microscopy.

Endpoints by digital reading were the same or within ± 1 dilution steps from that of manual reading for 100%, 60% and 90% of the cases at the three testing sites, and within ± 2 dilution steps for the rest of the samples.

{20}------------------------------------------------

| | | Site #1 | | | | Site #2 | | | | Site #3 | | | |
|--------|-----------|-------------------------------|-----------------------|---------|-------------------|-------------------------------|-----------------------|---------|-------------------|-------------------------------|-----------------------|---------|-------------------|
| | | Manual microscopic<br>reading | Digital image reading | | | Manual microscopic<br>reading | Digital image reading | | | Manual microscopic<br>reading | Digital image reading | | |
| Number | Sample ID | Pattern | Endpoint<br>titer | Pattern | Endpoint<br>titer | Pattern | Endpoint<br>titer | Pattern | Endpoint<br>titer | Pattern | Endpoint<br>titer | Pattern | Endpoint<br>titer |
| 1 | NVB095 | C | 1280 | C | 1280 | C | 640 | C | 640 | C | 640 | C | 320 |
| 2 | NVB113 | C | 640 | C | 640 | C | 640 | C | 640 | C | 320 | C | 160 |
| 3 | NVB020 | H | 640 | H | 320 | H | 160 | H | 160 | H | 320 | H | 320 |
| 4 | NVB071 | CS | 640 | S | 640 | S | 1280 | S | 320 | S | 1280 | S | 1280 |
| 5 | NVB074 | D | 320 | D | 640 | D | 320 | D | 640 | D | 320 | D | 160 |
| 6 | NVB056 | H | 1280 | H | 640 | H | 640 | H | 640 | H | 1280 | H | 640 |
| 7 | NVB042 | N | 2560 | N | 2560 | N | ≥5120 | N | 1280 | N | 2560 | N | 1280 |
| 8 | NVB014 | N | 1280 | N | 1280 | N | 2560 | N | 1280 | N | 1280 | N | 640 |
| 9 | NVB118 | H | 640 | H | 640 | H | 1280 | H | 320 | H | 1280 | H | 320 |
| 10 | NVB036 | H | 1280 | H | 1280 | H | 2560 | H | 640 | H | 1280 | H | 1280 |

Highlighted results: two dilution steps difference between manual and digital reading.

#### 18.2 Agreement on clinical sample cohort

To assess the agreement between NOVA View generated results obtained by reading the digital images and results obtained by manual reading of the slides, a study was performed at Inova Diagnostics (Site #1) and at two external locations (Site #2 and Site #3).

A cohort of 120 samples (same samples at each location) were processed with the NOVA Lite DAPI ANA kit, and read with NOVA View. The 120 samples were selected to represent approximately 50% negative and 50% positive samples with various patterns. All major patterns were represented, and reactivity grades ranged from 0 to 4. Digital images were interpreted and confirmed. Additionally, slides were read with traditional fluorescent microscope by the same operator. Results are presented below.

Agreement between manual reading, digital reading and NOVA View results:

| | | Positive Agrmnt %<br>(95% CI) | Negative Agrmnt %<br>(95% CI) | Total Agrmnt %<br>(95% CI) |
|---------|----------------------|-------------------------------|-------------------------------|----------------------------|
| Site #1 | Manual vs NOVA View | 100.0 (93.7-100.0) | 98.4 (91.5-100.0) | 99.2 (95.4-100.0) |
| | Manual vs digital | 100.0 (93.7-100.0) | 98.4 (91.5-100.0) | 99.2 (95.4-100.0) |
| | Digital vs NOVA View | 100.0 (93.8-100.0) | 100.0 (94.2-100.0) | 100.0 (97.0-100.0) |
| Site #2 | Manual vs NOVA View | 95.0 (81.6-99.0) | 98.3 (91.1-100.0) | 96.7 (91.7-99.1) |
| | Manual vs digital | 96.7 (88.5-99.6) | 95.0 (86.1-99.0) | 95.8 (90.5-98.6) |
| | Digital vs NOVA View | 93.4 (84.1-98.2) | 98.3 (90.9-100.0) | 95.8 (90.5-98.6) |
| Site #3 | Manual vs NOVA View | 94.6 (85.1-96.8) | 98.4 (91.6-100.0) | 96.7 (91.7-99.1) |
| | Manual vs digital | 92.9 (82.7-98.0) | 100. (94.4-100.0) | 96.7 (91.7-99.1) |

{21}------------------------------------------------

| | Digital vs NOVA View | 100.0 (93.2-100.0) | 97.1 (89.8-99.6) | 98.3 (94.1-99.8) |
|--|----------------------|--------------------|------------------|------------------|
|--|----------------------|--------------------|------------------|------------------|

### Pattern agreement

Pattern agreement was assessed in pair-wise comparison between manual reading, NOVA View results, and digital image reading at each site. Only definitive patterns (Homogeneous, Speckled, Centromere, Nucleolar, Nuclear dots) were considered as pattern agreement. NOVA View reported "Unrecognized" patterns and user reported "Other" patterns were not considered as an agreement.

Out of the 120 samples in the reproducibility cohort, there were 57 positive samples at Site#1, 60 at Site#2 and 56 at Site#3 by manual reading (reference method). Summary table of pattern agreement is shown below.

| Reproducibility cohort n=120 | Number (%) of samples with pattern agreement* | | | | |
|------------------------------|-----------------------------------------------|------------|------------|--|--|
| | Site#1 | Site#2 | Site#3 | | |
| Manual vs Digital | 55 (96.5%) | 57 (95.0%) | 54 (96.4%) | | |
| Manual vs NOVA View | 45 (78.9%) | 50 (83.3%) | 45 (80.4%) | | |
| Digital vs NOVA View | 44 (77.2%) | 48 (80.0%) | 45 (80.4%) | | |

*As percentage of samples that were positive with manual interpretation

### Grades correlation(agreement)

Fluorescence intensity grades were within ± one grade from each other between manual reading and digital image reading, as shown below:

| Reproducibility cohort n=120 | Percent of samples within ± one grade | | | | | | |
|------------------------------|---------------------------------------------|--|--|--|--|--|--|
| Manual vs digital | Site#1 Site#2 Site#3 98.30% 99.20% 99.20% | | | | | | |

#### Clinical performance 19.

#### 19.1 Clinical sensitivity and specificity

To assess clinical performance, a clinical study was performed at Inova Diagnostics (Site #1) and at two external sites (shown as Site #2 and Site #3).

{22}------------------------------------------------

A cohort of 463 clinically characterized samples (same samples at each location) were processed with NOVA Lite DAPI ANA kit, and read with NOVA View. Digital images were interpreted and confirmed. Additionally, slides were read with traditional manual microscope by the same operator.

The number and distribution of the samples are shown below:

| Sample type | Number of<br>samples |
|---------------------------------------|----------------------|
| Healthy control | 75 |
| HBV | 20 |
| HCV | 5 |
| HIV | 5 |
| Syphilis | 5 |
| SLE | 75 |
| SSc | 20 |
| SS | 20 |
| AIL | 20 |
| RA | 20 |
| MCTD | 21 |
| Autoimmune myositis | 26 |
| Fibromyalgia | 25 |
| Anti-MPO/anti-PR3 | 26 |
| Crohn's/Inflammatory<br>bowel disease | 20 |
| Autoimmune<br>thyroiditis | 24 |
| Celiac disease | 24 |
| Drug induced lupus | 25 |
| Other | 7 |
| Total | 463 |

{23}------------------------------------------------

Positivity rate in the various sample groups at the three locations is listed below:

### Site #1:

| | | Number of positive samples | | | | | % positive samples | | |
|------------------------------------|-------------------|----------------------------|-------------|--------------|------------------------------------|-------------------|--------------------|-------------|--------------|
| Sample type | Number of samples | NOVA View | Manual read | Digital read | Sample type | Number of samples | NOVA View | Manual read | Digital read |
| Healthy control | 75 | 4 | 4 | 5 | Healthy control | 75 | 5.3% | 5.3% | 6.7% |
| HBV | 20 | 1 | 5 | 3 | HBV | 20 | 5.0% | 25.0% | 15.0% |
| HCV | 5 | 2 | 0 | 2 | HCV | 5 | 40.0% | 0.0% | 40.0% |
| HIV | 5 | 2 | 0 | 2 | HIV | 5 | 40.0% | 0.0% | 40.0% |
| Syphilis | 5 | 3 | 0 | 3 | Syphilis | 5 | 60.0% | 0.0% | 60.0% |
| SLE | 75 | 60 | 54 | 60 | SLE | 75 | 80.0% | 72.0% | 80.0% |
| SSc | 20 | 19 | 19 | 19 | SSc | 20 | 95.0% | 95.0% | 95.0% |
| SS | 20 | 12 | 9 | 13 | SS | 20 | 60.0% | 45.0% | 65.0% |
| AIL | 20 | 20 | 16 | 20 | AIL | 20 | 100.0% | 80.0% | 100.0% |
| RA | 20 | 15 | 11 | 14 | RA | 20 | 75.0% | 55.0% | 70.0% |
| MCTD | 21 | 10 | 10 | 10 | MCTD | 21 | 47.6% | 47.6% | 47.6% |
| Autoimmune myositis | 26 | 6 | 7 | 6 | Autoimmune myositis | 26 | 23.1% | 26.9% | 23.1% |
| Fibromyalgia | 25 | 6 | 9 | 6 | Fibromyalgia | 25 | 24.0% | 36.0% | 24.0% |
| Anti-MPO/anti-PR3 | 26 | 1 | 3 | 4 | Anti-MPO/anti-PR3 | 26 | 3.8% | 11.5% | 15.4% |
| Crohn's/Inflammatory bowel disease | 20 | 8 | 9 | 8 | Crohn's/Inflammatory bowel disease | 20 | 40.0% | 45.0% | 40.0% |
| Autoimmune thyroiditis | 24 | 2 | 4 | 3 | Autoimmune thyroiditis | 24 | 8.3% | 16.7% | 12.5% |
| Celiac disease | 24 | 3 | 4 | 3 | Celiac disease | 24 | 12.5% | 16.7% | 12.5% |
| Drug induced lupus | 25 | 4 | 5 | 5 | Drug induced lupus | 25 | 16.0% | 20.0% | 20.0% |
| Other | 7 | 1 | 2 | 1 | Other | 7 | 14.3% | 28.6% | 14.3% |
| Total | 463 | 179 | 171 | 187 | Total | 463 | | | |

{24}------------------------------------------------

# NOVA Lite® DAPI ANA Kit

### Site #2:

| | Number of pos samples | | | | | | % positive samples | | |
|---------------------------------------|-----------------------|--------------|----------------|-----------------|-----------------------------------------|----------------------|--------------------|----------------|--------------|
| Sample type | Number of<br>samples | NOVA<br>View | Manual<br>read | Digital<br>read | Sample type | Number of<br>samples | NOVA View | Manual<br>read | Digital read |
| Healthy control | 75 | 2 | 7 | 2 | Healthy control | 75 | 2.7% | 9.3% | 2.7% |
| HBV | 20 | 1 | 4 | 3 | HBV | 20 | 5.0% | 20.0% | 15.0% |
| HCV | 5 | 1 | 1 | 1 | HCV | 5 | 20.0% | 20.0% | 20.0% |
| HIV | 5 | 2 | 0 | 1 | HIV | 5 | 40.0% | 0.0% | 20.0% |
| Syphilis | 5 | 1 | 0 | 0 | Syphilis | 5 | 20.0% | 0.0% | 0.0% |
| SLE | 75 | 54 | 53 | 55 | SLE | 75 | 72.0% | 70.7% | 73.3% |
| SSc | 20 | 19 | 19 | 19 | SSC | 20 | 95.0% | 95.0% | 95.0% |
| SS | 20 | 9 | 11 | 9 | SS | 20 | 45.0% | 55.0% | 45.0% |
| AIL | 20 | 17 | 18 | 17 | AIL | 20 | 85.0% | 90.0% | 85.0% |
| RA | 20 | 13 | 15 | 14 | RA | 20 | 65.0% | 75.0% | 70.0% |
| MCTD | 21 | 8 | 10 | 8 | MCTD | 21 | 38.1% | 47.6% | 38.1% |
| Autoimmune myositis | 26 | 8 | 9 | 7 | Autoimmune myositis | 26 | 30.8% | 34.6% | 26.9% |
| Fibromyalgia | 25 | 5 | 11 | 6 | Fibromyalgia | 25 | 20.0% | 44.0% | 24.0% |
| Anti-MPO/anti-PR3 | 26 | 5 | 5 | 4 | Anti-MPO/anti-PR3 | 26 | 19.2% | 19.2%…