NOVA Lite DAPI ANCA Ethanol Kit, NOVA Lite DAPI ANCA Formalin Kit

K161258 · Inova Diagnostics, Inc. · MOB · Feb 3, 2017 · Immunology

Device Facts

• Record ID: K161258
• Device Name: NOVA Lite DAPI ANCA Ethanol Kit, NOVA Lite DAPI ANCA Formalin Kit
• Applicant: Inova Diagnostics, Inc.
• Product Code: MOB · Immunology
• Decision Date: Feb 3, 2017
• Decision: SESE
• Submission Type: Traditional
• Regulation: 21 CFR 866.5660
• Device Class: Class 2

Indications for Use

NOVA Lite DAPI ANCA (Ethanol) Kit is an indirect immunofluorescence assay for the qualitative detection and semi-quantitative determination of anti-neutrophil cytoplasmic antibodies (ANCA) of IgG isotypes in human serum by manual fluorescence microscopy or with NOVA View Automated Fluorescence Microscope. The presence of ANCA, in conjunction with other serological, radiological, histological, and clinical findings, aids in the diagnosis of ANCA associated vasculitides. A trained operator must confirm results when generated with the NOVA View device. NOVA Lite DAPI ANCA (Formalin) Kit is an indirect immunofluorescence assay for the qualitative detection and semi-quantitative determination of anti-neutrophil cytoplasmic antibodies (ANCA) of IgG isotypes in human serum by manual fluorescence microscopy or with NOVA View Automated Fluorescence Microscope. The presence of ANCA, in conjunction with other serological, radiological, histological, and clinical findings aids in the diagnosis of ANCA associated vasculitides. A trained operator must confirm results when generated with the NOVA View device. ANCA Formalin test is not intended to be used by itself, but in conjunction with ANCA Ethanol test.

Device Story

Indirect immunofluorescence assay (IFA) using human neutrophil granulocytes fixed on glass slides (ethanol or formalin). Patient serum is incubated with substrate; unbound antibodies washed; then incubated with FITC-conjugated anti-human IgG containing DAPI (DNA-binding blue fluorescent dye). Slides are read via manual fluorescence microscopy or scanned by NOVA View Automated Fluorescence Microscope. NOVA View captures digital images, measures FITC light intensity (LIU), and provides classification/pattern information. Results are reviewed by a trained operator on a computer monitor. Aids in diagnosis of ANCA-associated vasculitides by identifying C-ANCA or P-ANCA patterns. Benefits include standardized digital image capture and automated intensity measurement, reducing subjectivity in interpretation.

Clinical Evidence

Clinical performance evaluated on 653 samples (including AAV, autoimmune, infectious, and healthy controls). Sensitivity and specificity calculated across manual, digital, and automated (NOVA View) interpretation methods. Between-site reproducibility study (n=287) showed high agreement across sites and operators. Method comparison to predicate (n=267) demonstrated acceptable qualitative, grade, and pattern agreement. Analytical performance included interference testing (up to 100 mg/dL bilirubin, 200 mg/dL hemoglobin, etc.) and cross-reactivity studies with ANA-positive samples.

Technological Characteristics

Indirect immunofluorescence assay. Substrate: Human neutrophil granulocytes on 12-well glass slides (ethanol or formalin fixed). Conjugate: FITC-conjugated anti-human IgG (Fc specific) with DAPI. Energy source: Fluorescence excitation. Connectivity: Networked/standalone via NOVA View system. Software: Automated image acquisition and analysis. Storage: 2-8°C. Shelf life: 18 months.

Indications for Use

Indicated for qualitative detection and semi-quantitative determination of IgG anti-neutrophil cytoplasmic antibodies (ANCA) in human serum to aid in the diagnosis of ANCA-associated vasculitides. For use by trained operators via manual fluorescence microscopy or the NOVA View Automated Fluorescence Microscope. Formalin kit must be used in conjunction with the Ethanol kit.

Regulatory Classification

Identification

A multiple autoantibodies immunological test system is a device that consists of the reagents used to measure by immunochemical techniques the autoantibodies (antibodies produced against the body's own tissues) in serum and other body fluids. Measurement of multiple autoantibodies aids in the diagnosis of autoimmune disorders (disease produced when the body's own tissues are injured by autoantibodies).

Predicate Devices

• NOVA Lite ANCA (K961340)

Reference Devices

• NOVA View Automated Fluorescence Microscope (K153150)

Submission Summary (Full Text)

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# 510(k) SUBSTANTIAL EQUIVALENCE DETERMINATION DECISION MEMORANDUM

A. 510(k) Number:
K161258

B. Purpose for Submission:
New Device

C. Measurand:
Anti-Neutrophil Cytoplasmic Antibodies (ANCA)

D. Type of Test:
Qualitative and/or semi-quantitative indirect immunofluorescence (IIF) assays

E. Applicant:
Inova Diagnostics, Inc.

F. Proprietary and Established Names:
NOVA Lite DAPI ANCA (Ethanol) Kit
NOVA Lite DAPI ANCA (Formalin) Kit

G. Regulatory Information:

1. Regulation section:
21 CFR §866.5660, Multiple autoantibodies immunological test system

2. Classification:
Class II

3. Product code:
MOB, Aneutrophil cytoplasmic antibodies (ANCA)

4. Panel:
Immunology (82)

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H. Intended Use:

1. Intended use(s):

NOVA Lite DAPI ANCA (Ethanol) Kit is an indirect immunofluorescence assay for the qualitative detection and semi-quantitative determination of anti-neutrophil cytoplasmic antibodies (ANCA) of IgG isotypes in human serum by manual fluorescence microscopy or with NOVA View Automated Fluorescence Microscope. The presence of ANCA, in conjunction with other serological, radiological, histological, and clinical findings, aids in the diagnosis of ANCA associated vasculitides. A trained operator must confirm results when generated with the NOVA View device.

NOVA Lite DAPI ANCA (Formalin) Kit is an indirect immunofluorescence assay for the qualitative detection and semi-quantitative determination of anti-neutrophil cytoplasmic antibodies (ANCA) of IgG isotypes in human serum by manual fluorescence microscopy or with NOVA View Automated Fluorescence Microscope. The presence of ANCA, in conjunction with other serological, radiological, histological, and clinical findings aids in the diagnosis of ANCA associated vasculitides. A trained operator must confirm results when generated with the NOVA View device. ANCA Formalin test is not intended to be used by itself, but in conjunction with ANCA Ethanol test.

2. Indication(s) for use:

Same as intended use.

3. Special conditions for use statement(s):

1. For prescription use only.
2. The device is for use by a trained operator in a clinical laboratory setting.
3. All software-aided results must be confirmed by the trained operator.

Special instrument requirements:

NOVA View Automated Fluorescence Microscope (cleared in K153150) or Olympus CX31 with a FRAEN LED (Blue 480nm) or equivalent microscope.

I. Device Description:

NOVA Lite DAPI ANCA (Ethanol) and DAPI ANCA (Formalin) Kits are indirect immunofluorescence assays for the qualitative detection and semi-quantitative determination of anti-neutrophil cytoplasmic antibodies of IgG isotypes in human serum.

Kit components include:

- ANCA (Formalin Fixed Human Neutrophils) Slides; 12 wells/slide, with desiccant, or ANCA (Ethanol Fixed Human Neutrophils) Slides; 12 wells/slide, with desiccant
- FITC IgG Conjugate with DAPI, containing 0.09% sodium azide, ready to use
- Positive Controls: cANCA and pANCA; human serum with antibodies to proteinase 3

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(PR3) and myeloperoxidase (MPO) antigen, containing 0.09% sodium azide; pre-diluted, ready to use.

- Negative Control: IFA System Negative Control, diluted human serum with no ANCA 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

Not provided in kit:

NOVA View Automated Fluorescence Microscope (cleared in K153150) or Olympus CX31 with a FRAEN LED (Blue 480nm) or equivalent microscope

## J. Substantial Equivalence Information:

1. Predicate device name:

NOVA Lite ANCA

2. Predicate 510(k) number:

K961340

3. Comparison with predicate:

| Similarities | | |
| --- | --- | --- |
| Item | Candidate | Predicate |
| Intended use | NOVA Lite® DAPI ANCA (Ethanol) Kit is an indirect immunofluorescence assay for the qualitative detection and semi-quantitative determination of anti-neutrophil cytoplasmic antibodies (ANCA) of IgG isotypes in human serum by manual fluorescence microscopy or with NOVA View Automated Fluorescence Microscope. The presence of ANCA, in conjunction with other serological tests and clinical findings aids in the assessment of ANCA associated vasculitides. A trained operator must confirm results when generated with the NOVA View device.
NOVA Lite® DAPI ANCA (Formalin) Kit is an indirect immunofluorescence | An indirect immunofluorescent test system utilizing ethanol-fixed human neutrophil substrate slides for detection of anti-neutrophil cytoplasmic antibodies (ANCA) in human serum. Detection of ANCA when used in conjunction with other laboratory and clinical findings aids in the assessment of various systemic vasculitides, such as Wegener's granulomatosis, microscopic polyarteritis and crescentic glomerulonephritis. |

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| Similarities | | |
| --- | --- | --- |
| Item | Candidate | Predicate |
| | assay for the qualitative detection and semi-quantitative determination of anti-neutrophil cytoplasmic antibodies (ANCA) of IgG isotypes in human serum by manual fluorescence microscopy or with NOVA View Automated Fluorescence Microscope. The presence of ANCA, in conjunction with other serological tests and clinical findings aids in the assessment of ANCA associated vasculitides. A trained operator must confirm results when generated with the NOVA View device. ANCA Formalin test is not intended to be used by itself, but in conjunction with ANCA Ethanol test. | |
| Reported Result | Qualitative and semiquantitative | Semi-quantitative |
| Analyte | Anti-neutrophil cytoplasmic antibodies of IgG isotype | Same |
| Methodology | Indirect immunofluorescence assay | Same |
| Manual Interpretation | Manual fluorescence microscopy | Same |
| Antigen | Human neutrophil granulocytes, on 12-well slides, ethanol fixed or formalin fixed. | Same |
| Sample Matrix | Serum | Same |
| Slides | 12-well coated with antigen | Same |
| Sample dilution | 1:20 | Same |
| Conjugate | FITC conjugated anti-human IgG (Fc specific) | Same |
| Controls | Positive Controls: cANCA and pANCA; human serum with antibodies to proteinase 3 (PR3) and myeloperoxidase (MPO) antigen.
Negative Control: IFA System
Negative Control, diluted human serum with no ANCA present. | Same |
| Storage | 2–8°C | Same |
| Shelf life | 18 months | Same |

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| Differences | | |
| --- | --- | --- |
| Item | Device | Predicate |
| Automated Interpretation | NOVA View® Automated Fluorescence Microscope | Manual only |
| Additional dye in Conjugate | 4',6-diamidino-2-phenylindole (DAPI) | None |

## K. Standard/Guidance Document Referenced:

1. Evaluation of Precision Performance of Quantitative Measurement Methods (CLSI EP05-A3)
2. Interference Testing in Clinical Chemistry (CLSI EP07-A2)
3. Method Comparison and Bias Estimation Using Patient Samples (CLSI EP09-A3)

## L. Test Principle:

Samples are diluted 1:20 in PBS and incubated with the antigen substrate (human neutrophil granulocytes fixed on glass microscope slides). After incubation, unbound antibodies are washed off. The substrate is then incubated with anti-human IgG-FITC conjugate. The conjugate contains the DNA-binding blue fluorescent dye, 4',6-diamidino-2-phenylindole (DAPI) that is required for NOVA View® Automated Fluorescence Microscope use. The blue dye is not visible by traditional fluorescence 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 scanned with the NOVA View Automated Fluorescence Microscope. The resulting digital images are reviewed and interpreted from the computer monitor. ANCA positive samples exhibit an apple green fluorescence corresponding to areas of the cell where autoantibody has bound.

### Manual interpretation of NOVA Lite DAPI ANCA (Ethanol) Kit:

The two major patterns on ethanol-fixed substrate are cytoplasmic or C-ANCA, and perinuclear or P-ANCA.

C-ANCA: C-ANCA samples present as coarse speckled cytoplasmic fluorescence, often with accentuated staining between the nuclear lobes. This pattern is characteristic for antibodies reacting with PR3.

P-ANCA: P-ANCA samples present as perinuclear staining with or without nuclear extension. This pattern is usually characteristic for antibodies reacting with MPO.

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Anti-nuclear antibody (ANA) positive samples, primarily those containing anti-DNA/histones, usually react with the nuclei of ethanol-fixed neutrophils, causing nuclear staining, and may mask or mimic the P-ANCA pattern(s). Moreover, antibodies to other granule antigens, such as elastase, BPI, cathepsin G, lactoferrin and others may also produce a perinuclear staining on ethanol-fixed neutrophil substrate (sometimes referred to as Atypical ANCA). Atypical ANCA and ANA usually become negative on formalin fixed substrate.

Manual interpretation of NOVA Lite DAPI ANCA (Formalin) Kit:

On formalin fixed substrate, both MPO and PR3 antibodies appear as coarse cytoplasmic granular staining with interlobular accentuation. Formalin fixation also destroys/modifies most of the nuclear antigens, and as a result, ANA positive samples usually become negative, or show greatly reduced fluorescence.

NOVA View® Automated Fluorescence Microscope interpretation of NOVA Lite DAPI ANCA (Ethanol) Kit and NOVA Lite DAPI ANCA (Formalin) Kit:

When slides are analyzed by NOVA View® Automated Fluorescence Microscope, digital images of representative fields of view of the well are captured. These digital images must be reviewed and interpreted from the computer monitor by a trained operator. At the same time when digital images are taken, NOVA View® Automated Fluorescence Microscope measures the FITC light intensity of the cells that are included in the region. NOVA View® Automated Fluorescence Microscope reports the measured fluorescence intensity in units of Light Intensity Units (LIU). NOVA View® Automated Fluorescence Microscope provides the trained operator with the acquired digital images and the following supportive information:

LIU value
- Suggested negative/positive classification
- Suggested pattern for NOVA Lite DAPI ANCA (Ethanol) Kit pattern nomenclature:

| P | P-ANCA |
| --- | --- |
| C | C-ANCA |
| U | Unrecognized |

- Suggested pattern NOVA Lite DAPI ANCA (Formalin) Kit pattern nomenclature:

| N | Nuclear |
| --- | --- |
| C | Cytoplasmic |
| U | Unrecognized |

# Software principle:

IFA fluorescence microscopy digital images are taken by a camera and stored on the computer system. All images are taken through a DAPI and a FITC filter. FITC (fluorescein isothiocyanate) is a green fluorescent dye that is chemically bound to the anti-human IgG conjugate. DAPI $(4^{\prime},6$ -diamidino-2-phenylindole) is a blue fluorescent dye added to the

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assay conjugate. DAPI strongly binds to DNA; therefore, it stains cell nuclei, regardless of antibody and conjugate binding. The NOVA View® Automated Fluorescence Microscope software is capable of storing and managing the digital images, including retrieving, displaying on computer monitor, enlarging, and overlaying DAPI and FITC images. At the same time when digital images are taken, the software measures FITC light intensity of the cells that are included in the region, and reports the measured fluorescence intensity. An assay specific software algorithm provides pattern recognition. The software includes a Single Well Titer (SWT) application that estimates the endpoint titer (e.g., the highest dilution that gives positive result) for wells with a positive reaction with Ethanol ANCA, based on the obtained fluorescence intensity and pattern.

## M. Performance Characteristics:

### 1. Analytical performance:

Nomenclature used in the studies:

- All studies have been performed by interpreting the results with both manual microscopy and with the NOVA View® Automated Fluorescence Microscope system.
- “Manual” and “Manual reading” refers to results obtained by reading the slides with a traditional fluorescence microscope.
- “NOVA View” refers to software reported results obtained with the NOVA View Automated Fluorescence Microscope, such as LIU, positive/negative classification and pattern information.
- “Digital”, “Digital reading” and “Digital image” refers to results obtained by reading NOVA View® Automated Fluorescence Microscope generated images on the computer monitor.
- Intensity of the staining is expressed in reactivity grades. Grade 0 is negative; grades 1–4 are weak to strong positive:
- 1+ Lowest specific fluorescence that enables the nuclear and/or cytoplasmic staining to be clearly differentiated from the background fluorescence
- 2+ Clearly distinguishable positive fluorescence
- 3+ Bright apple green fluorescence
- 4+ Brilliant apple green fluorescence

For all studies presented here, all results met the sponsor’s pre-determined acceptance criteria.

### a. Precision/Reproducibility:

#### Within-Lab Precision:

To assess the precision performance of the NOVA Lite DAPI ANCA (Ethanol) and ANCA (Formalin) Kit results, a study was performed by processing three negative and 13 positive samples: seven anti-MPO and six anti-PR3 positive samples with various ANCA intensities, in three replicates, in ten runs, two runs per day, resulting

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in 30 data points for each sample. The slides were read with NOVA View® Automated Fluorescence Microscope, and digital images were interpreted by one operator. Additionally, slides were read with a manual microscope. Three set of results were generated: NOVA View® Automated Fluorescence Microscope software interpretation, digital image reading results and manual reading results.

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. Results are presented in the table below:

NOVA Lite DAPI ANCA (Ethanol) Kit qualitative results repeatability and reproducibility

| | | | | NOVA View® Automated Fluorescence Microscope results | | | Manual results | | Digital results | |
| --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- |
| Sample ID | Specificity | N | Mean LIU | Pattern | % Negative | % Positive | % Negative | % Positive | % Negative | % Positive |
| 1 | Anti-MPO | 30 | 1380.7 | P | 0 | 100 | 0 | 100 | 0 | 100 |
| 14 | Anti-MPO | 30 | 850.8 | P | 0 | 100 | 0 | 100 | 0 | 100 |
| 12 | Anti-MPO | 30 | 658.8 | P | 0 | 100 | 0 | 100 | 0 | 100 |
| 13 | Anti-MPO | 30 | 563.3 | P | 0 | 100 | 0 | 100 | 0 | 100 |
| 2 | Anti-MPO | 30 | 424.9 | P | 0 | 100 | 0 | 100 | 0 | 100 |
| 3 | Anti-MPO | 30 | 341.9 | P | 0 | 100 | 0 | 100 | 0 | 100 |
| 4 | Anti-MPO | 30 | 74.5 | P | 0 | 100 | 0 | 100 | 0 | 100 |
| 17 | Anti-MPO | 30 | 36.4 | P | 13 | 87 | 0 | 100 | 7 | 93 |
| 20 | Anti-MPO | 30 | 18.7 | P | 63 | 37 | 0 | 100 | 23 | 77 |
| 26 | Anti-MPO | 30 | 16.0 | P | 70 | 30 | 0 | 100 | 40 | 60 |
| 25 | Anti-MPO | 30 | 15.1 | P | 77 | 23 | 0 | 100 | 30 | 70 |
| 18 | Anti-MPO | 30 | 13.1 | P | 87 | 13 | 0 | 100 | 40 | 60 |
| 5 | N/A | 30 | 2.0 | - | 100 | 0 | 100 | 0 | 100 | 0 |
| 6 | Anti-PR3 | 30 | 1056.6 | C | 0 | 100 | 0 | 100 | 0 | 100 |
| 15 | Anti-PR3 | 30 | 866.1 | C | 0 | 100 | 0 | 100 | 0 | 100 |
| 7 | Anti-PR3 | 30 | 388.2 | C | 0 | 100 | 0 | 100 | 0 | 100 |
| 8 | Anti-PR3 | 30 | 80.0 | C | 0 | 100 | 0 | 100 | 0 | 100 |
| 9 | Anti-PR3 | 30 | 34.4 | C | 13 | 87 | 0 | 100 | 0 | 100 |
| 21 | Anti-PR3 | 30 | 70.0 | C | 0 | 100 | 0 | 100 | 0 | 100 |
| 23 | Anti-PR3 | 30 | 57.1 | C | 23 | 77 | 0 | 100 | 10 | 90 |
| 22 | Anti-PR3 | 30 | 57.0 | C | 13 | 87 | 0 | 100 | 0 | 100 |
| 19 | Anti-PR3 | 30 | 29.9 | C | 30 | 70 | 0 | 100 | 0 | 100 |
| 24 | Anti-PR3 | 30 | 26.0 | C | 33 | 67 | 0 | 100 | 13 | 87 |
| 10 | Anti-PR3 | 30 | 3.4 | - | 100 | 0 | 100 | 0 | 100 | 0 |
| 11 | N/A | 30 | 2.3 | - | 100 | 0 | 100 | 0 | 100 | 0 |
| 16 | N/A | 30 | 2.0 | - | 100 | 0 | 100 | 0 | 100 | 0 |

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NOVA Lite DAPI ANCA (Formalin) Kit qualitative results repeatability and reproducibility

| | | | | NOVA View® Automated Fluorescence Microscope results | | | Manual results | | Digital results | |
| --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- |
| Sample ID | Specificity | N | Mean LIU | Pattern | % Negative | % Positive | % Negative | % Positive | % Negative | % Positive |
| 1 | Anti-MPO | 30 | 788.8 | C | 0 | 100 | 0 | 100 | 0 | 100 |
| 14 | Anti-MPO | 30 | 517.7 | C | 0 | 100 | 0 | 100 | 0 | 100 |
| 12 | Anti-MPO | 30 | 406.7 | C | 0 | 100 | 0 | 100 | 0 | 100 |
| 13 | Anti-MPO | 30 | 263.8 | C | 0 | 100 | 0 | 100 | 0 | 100 |
| 2 | Anti-MPO | 30 | 218.7 | C | 0 | 100 | 0 | 100 | 0 | 100 |
| 3 | Anti-MPO | 30 | 127.4 | C | 0 | 100 | 0 | 100 | 0 | 100 |
| 4 | Anti-MPO | 30 | 17.4 | C/- | 93 | 7 | 0 | 100 | 0 | 100 |
| 17 | Anti-MPO | 30 | 28.8 | C | 80 | 20 | 0 | 100 | 17 | 83 |
| 20 | Anti-MPO | 30 | 36.7 | C | 30 | 70 | 0 | 100 | 0 | 100 |
| 26 | Anti-MPO | 30 | 35.7 | C | 37 | 63 | 0 | 100 | 3 | 97 |
| 25 | Anti-MPO | 30 | 34 | C | 50 | 50 | 0 | 100 | 10 | 90 |
| 18 | Anti-MPO | 30 | 32.5 | C | 43 | 57 | 0 | 100 | 3 | 97 |
| 5 | N/A | 30 | 2.0 | - | 100 | 0 | 100 | 0 | 100 | 0 |
| 6 | Anti-PR3 | 30 | 1104.9 | C | 0 | 100 | 0 | 100 | 0 | 100 |
| 15 | Anti-PR3 | 30 | 1095.0 | C | 0 | 100 | 0 | 100 | 0 | 100 |
| 7 | Anti-PR3 | 30 | 491.2 | C | 0 | 100 | 0 | 100 | 0 | 100 |
| 8 | Anti-PR3 | 30 | 131.4 | C | 0 | 100 | 0 | 100 | 0 | 100 |
| 9 | Anti-PR3 | 30 | 128.5 | C | 0 | 100 | 0 | 100 | 0 | 100 |
| 21 | Anti-PR3 | 30 | 22.9 | C | 77 | 23 | 0 | 100 | 13 | 87 |
| 23 | Anti-PR3 | 30 | 26.9 | C | 70 | 30 | 0 | 100 | 13 | 87 |
| 22 | Anti-PR3 | 30 | 24.3 | C | 87 | 13 | 0 | 100 | 3 | 97 |
| 19 | Anti-PR3 | 30 | 16.8 | C | 90 | 10 | 0 | 100 | 57 | 43 |
| 24 | Anti-PR3 | 30 | 20.3 | C | 87 | 13 | 0 | 100 | 17 | 83 |
| 10 | Anti-PR3 | 30 | 2.8 | - | 100 | 0 | 100 | 0 | 100 | 0 |
| 11 | N/A | 30 | 2.0 | - | 100 | 0 | 100 | 0 | 100 | 0 |
| 16 | N/A | 30 | 2.0 | - | 100 | 0 | 100 | 0 | 100 | 0 |

Between site reproducibility:

The same 287 clinically-characterized samples were tested with both NOVA Lite DAPI ANCA (Ethanol) Kit and NOVA Lite DAPI ANCA (Formalin) Kit at three testing sites, using three different NOVA View® Automated Fluorescence Microscope instruments: in Inova's laboratory, and two external US clinical

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laboratories. The two external sites each also tested 100 routine clinical samples that were submitted for ANCA testing. The composition of the sample cohorts included patients with ANCA associated vasculitis (AAV) and a number of autoimmune and infectious diseases. The results obtained by the three sites are shown below:

NOVA Lite DAPI ANCA (Ethanol) Kit:

Manual Reading Between-Site:

| Manual Reading N=287 | Site #1 vs. Site #2 | Site #1 vs. Site #3 | Site #2 vs. Site #3 |
| --- | --- | --- | --- |
| Average Positive Agreement % (95%CI) | 80.8 (74.8–85.7) | 84.5 (78.8–88.9) | 95.6 (91.2–97.9) |
| Average Negative Agreement % (95%CI) | 98.9 (93.9–99.8) | 94.4 (87.5–97.6) | 86.3 (79.1–91.3) |
| Overall Agreement % (95%CI) | 86.4 (82.0–89.9) | 87.6 (83.3–91.0) | 91.5 (87.7–94.2) |

Digital Reading Between-Site:

| Digital Reading* N=287 | Site #1 vs. Site #2 | Site #1 vs. Site #3 | Site #2 vs. Site #3 |
| --- | --- | --- | --- |
| Average Positive Agreement % (95%CI) | 85.5 (79.7–89.8) | 84.7 (78.8–89.2) | 96.2 (91.9–98.2) |
| Average Negative Agreement % (95%CI) | 100 (96.3–100) | 100 (96.3–100) | 96.1 (91.1–98.3) |
| Overall Agreement % (95%CI) | 90.6 (86.7–93.5) | 90.1 (86.1–93.1) | 96.1 (93.2–97.8) |

NOVA View® Automated Fluorescence Microscope Interpretation Between-Site:

| NOVA View® Automated Fluorescence MicroscopeN=287 | Site #1 vs. Site #2 | Site #1 vs. Site #3 | Site #2 vs. Site #3 |
| --- | --- | --- | --- |
| Average Positive Agreement % (95%CI) | 86.2 (80.3–90.6) | 86.0 (80.1–90.4) | 96.0 (91.5–98.2) |
| Average Negative Agreement % (95%CI) | 98.2 (93.8–99.5) | 99.1 (95.1–99.8) | 96.2 (91.5–98.4) |
| Overall Agreement % (95%CI) | 90.9 (87.1–93.7) | 91.2 (87.3–93.9) | 96.1 (93.2–97.8) |

Grade Agreement:

| Manual Reading N=287 | Within ± 1 grade |
| --- | --- |
| Site 1 vs Site 2 | 98.0% |
| Site 1 vs Site 3 | 96.0% |
| Site 2 vs Site 3 | 96.0% |

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| Digital Reading N=287 | Within ± 1 grade |
| --- | --- |
| Site 1 vs Site 2 | 98.0% |
| Site 1 vs Site 3 | 97.0% |
| Site 2 vs Site 3 | 99.0% |

Pattern Agreement:

Qualitative pattern agreement was assessed between sites.

| NOVA View® Automated Fluorescence Microscope N=287 | Pattern Agreement with positive/negative disagreement included |
| --- | --- |
| Site 1 vs Site 2 | 84.0% |
| Site 1 vs Site 3 | 81.0% |
| Site 2 vs Site 3 | 90.0% |
| Digital N=287 | Pattern Agreement with positive/negative disagreement included |
| --- | --- |
| Site 1 vs Site 2 | 89.0% |
| Site 1 vs Site 3 | 89.0% |
| Site 2 vs Site 3 | 96.0% |
| Manual N=287 | Pattern Agreement with positive/negative disagreement included |
| --- | --- |
| Site 1 vs Site 2 | 84.0% |
| Site 1 vs Site 3 | 86.0% |
| Site 2 vs Site 3 | 92.0% |

NOVA Lite DAPI ANCA (Formalin) Kit:

Manual Reading Between-Site:

| Manual Reading N=287 | Site #1 vs. Site #2 | Site #1 vs. Site #3 | Site #2 vs. Site #3 |
| --- | --- | --- | --- |
| Average Positive Agreement % (95%CI) | 81.9 (74.6–87.4) | 86.2 (79.5–91.0) | 92.2 (85.9–95.9) |
| Average Negative Agreement % (95%CI) | 98.0 (94.2–99.3) | 96.0 (91.5–98.1) | 89.5 (84.0–93.2) |
| Overall Agreement % (95%CI) | 90.2 (86.3–93.2) | 91.3 (87.5–94.0) | 90.6 (86.7–93.5) |

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Digital Reading Between-Site:

| Digital Reading* N=287 | Site #1 vs. Site #2 | Site #1 vs. Site #3 | Site #2 vs. Site #3 |
| --- | --- | --- | --- |
| Average Positive Agreement % (95%CI) | 85.5 (78.5–90.5) | 90.8 (84.7–94.7) | 97.4 (92.5–99.1) |
| Average Negative Agreement % (95%CI) | 98.7 (95.4–99.6) | 98.7 (95.4–99.6) | 94.2 (89.7–96.8) |
| Overall Agreement % (95%CI) | 92.7 (89.1–95.2) | 95.1 (92.0–97.1) | 95.5 (92.4–97.3) |

NOVA View® Automated Fluorescence Microscope Interpretation Between-Site:

| NOVA View® Automated Fluorescence Microscope N=287 | Site #1 vs. Site #2 | Site #1 vs. Site #3 | Site #2 vs. Site #3 |
| --- | --- | --- | --- |
| Average Positive Agreement % (95%CI) | 88.8 (81.8–93.3) | 92.2 (85.9–95.9) | 96.3 (90.9–98.6) |
| Average Negative Agreement % (95%CI) | 97.1 (93.3–98.7) | 95.3 (91.0–97.6) | 93.9 (89.3–96.5) |
| Overall Agreement % (95%CI) | 93.7 (90.3–96.0) | 94.1 (90.7–96.3) | 94.8 (91.6–96.8) |

Grade Agreement

| Manual Reading N=287 | Within ± 1 grade |
| --- | --- |
| Site 1 vs Site 2 | 98.0% |
| Site 1 vs Site 3 | 94.0% |
| Site 2 vs Site 3 | 91.0% |
| Digital Reading N=287 | Within ± 1 grade |
| Site 1 vs Site 2 | 99.0% |
| Site 1 vs Site 3 | 99.0% |
| Site 2 vs Site 3 | 98.0% |

Pattern Agreement

| NOVA View® Automated Fluorescence Microscope N=287 | Pattern Agreement with positive/negative disagreement included |
| --- | --- |
| Site 1 vs Site 2 | 87.0% |
| Site 1 vs Site 3 | 88.0% |
| Site 2 vs Site 3 | 89.0% |

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| Digital N=287 | Pattern Agreement with positive/negative disagreement included |
| --- | --- |
| Site 1 vs Site 2 | 92.0% |
| Site 1 vs Site 3 | 94.0% |
| Site 2 vs Site 3 | 94.0% |
| Manual N=287 | Pattern Agreement with positive/negative disagreement included |
| --- | --- |
| Site 1 vs Site 2 | 90.0% |
| Site 1 vs Site 3 | 90.0% |
| Site 2 vs Site 3 | 90.0% |

# Between-Operator Agreement:

Ten native clinical samples from patients with AAV and controls were selected to include two negative, four P-ANCA positive, and four C-ANCA positive samples. These samples were tested at each site for five days in five replicates (25 data points per sample). Manual and digital reading was performed by two operators at each site. Operator-to-operator reproducibility was assessed within each site and is presented below as overall agreement.

Operator-to-Operator Reproducibility: NOVA Lite DAPI ANCA (Ethanol) Kit

| | Percent Overall Agreement | | |
| --- | --- | --- | --- |
| | Site 1 | Site 2 | Site 3 |
| Manual Reading | 100% | 100% | 94.4% |
| Digital Reading | 100% | 97.2% | 98.8% |

Operator-to-Operator Reproducibility: NOVA Lite DAPI ANCA (Formalin) Kit

| | Percent Overall Agreement | | |
| --- | --- | --- | --- |
| | Site 1 | Site 2 | Site 3 |
| Manual Reading | 99.2% | 100% | 94.0 |
| Digital Reading | 97.2% | 99.6% | 91.6% |

The percent CVs demonstrated acceptable operator-to-operator reproducibility.

# Lot-to-Lot Comparison:

A lot-to-lot reproducibility study was also performed using three reagent lots each for the ethanol and formalin reagents. Thirty three sera were tested.

The following comparisons were made:

Digital image reading: qualitative agreement, grade and pattern agreement

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- Manual image reading: qualitative agreement, grade and pattern agreement
- NOVA View® Automated Fluorescence Microscope output: qualitative agreement and pattern agreement.

Lot-to-Lot, NOVA Lite DAPI ANCA (Ethanol) Kit: Qualitative Agreement:

NOVA View® Automated Fluorescence Microscope Reading Between-Lot Agreement:

| NOVA View® Automated Fluorescence Microscope Reading | % Negative Agreement (95%CI) | % Positive Agreement (95%CI) | %Total Agreement |
| --- | --- | --- | --- |
| Lot 017103 vs lot 018119 | 100.0 (61.0–100.0) | 96.3 (81.7–99.3) | 97.0 (84.7–99.5) |
| Lot 017103 vs lot 022657 | 100.0 (61.0–100.0) | 96.3 (81.7–99.3) | 97.0 (84.7–99.5) |
| Lot 018119 vs lot 022657 | 100.0 (64.6–100.0) | 100.0 (87.1–100.0) | 100.0 (89.6–100.0) |

Digital Reading Between-Lot Agreement:

| Digital Reading | % Negative Agreement (95%CI) | % Positive Agreement (95%CI) | %Total Agreement |
| --- | --- | --- | --- |
| Lot 017103 vs lot 018119 | 100.0 (61.0–100.0) | 100.0 (87.5–100.0) | 100.0 (89.6 -100.0) |
| Lot 017103 vs lot 022657 | 100.0 (61.0–100.0) | 100.0 (87.5–100.0) | 100.0 (89.6 -100.0) |
| Lot 018119 vs lot 022657 | 100.0 (61.0–100.0) | 100.0 (87.5–100.0) | 100.0 (89.6 -100.0) |

Manual Reading Between-Lot Agreement:

| Manual Reading | % Negative Agreement (95%CI) | % Positive Agreement (95%CI) | %Total Agreement |
| --- | --- | --- | --- |
| Lot 017103 vs lot 018119 | 100.0 (61.0–100.0) | 100.0 (87.5–100.0) | 100.0 (89.6 -100.0) |
| Lot 017103 vs lot 022657 | 100.0 (61.0–100.0) | 100.0 (87.5–100.0) | 100.0 (89.6 -100.0) |
| Lot 018119 vs lot 022657 | 100.0 (61.0–100.0) | 100.0 (87.5–100.0) | 100.0 (89.6 -100.0) |

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Lot-to-Lot, NOVA Lite DAPI ANCA (Formalin) Kit: Qualitative Agreement:

NOVA View® Automated Fluorescence Microscope Reading Between-Lot Agreement:

| NOVA View® Automated Fluorescence Microscope Reading | % Negative Agreement (95%CI) | % Positive Agreement (95%CI) | % Total Agreement |
| --- | --- | --- | --- |
| Lot 018714 vs Lot 024112 | 100.0 (74.1–100.0) | 95.5 (78.2–99.2) | 97.0 (84.7–99.5) |
| Lot 018714 vs Lot RP0002 | 81.8 (52.3–94.9) | 95.5 (78.2–99.2) | 90.9 (76.4–96.9) |
| Lot 024112 vs Lot RP0002 | 83.3 (55.2–95.3) | 100.0 (84.5–100.0) | 93.9 (80.4–98.3) |

Digital Reading Between-Lot Agreement:

| Digital Reading | % Negative Agreement (95%CI) | % Positive Agreement (95%CI) | % Total Agreement |
| --- | --- | --- | --- |
| Lot 018714 vs Lot 024112 | 100.0 (70.1–100.0) | 100.0 (86.2 -100.0) | 100.0 (89.6–100.0) |
| Lot 018714 vs Lot RP0002 | 77.8 (45.3–93.7) | 95.8 (79.8–99.3) | 90.9 (76.4–96.9) |
| Lot 024112 vs Lot RP0002 | 77.8 (45.3–93.7) | 95.8 (79.8–99.3) | 90.9 (76.4–96.9) |

Manual Reading Between-Lot Agreement:

| Manual Reading | % Negative Agreement (95%CI) | % Positive Agreement (95%CI) | % Total Agreement |
| --- | --- | --- | --- |
| Lot 018714 vs Lot 024112 | 100.0 (64.6–100.0) | 100.0 (87.1–100.0) | 100.0 (89.6–100.0) |
| Lot 018714 vs Lot RP0002 | 100.0 (64.6–100.0) | 100.0 (87.1–100.0) | 100.0 (89.6–100.0) |
| Lot 024112 vs Lot RP0002 | 100.0 (87.1–100.0) | 100.0 (87.1–100.0) | 100.0 (89.6–100.0) |

Conclusion:

Total agreement for ethanol slides ranged from 97.0% to 100.0% and was acceptable. Total agreement for formalin slides ranged from 90.9% to 100.0% and was acceptable.

Lot-to-Lot Grade Agreement:

All grades (100%) were within ± 1 grade from each other for all samples in all pair-

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wise comparisons between lots, on both ethanol and formalin slides.

## Lot-to-Lot Pattern Agreement:

**Pattern agreement, NOVA Lite DAPI ANCA (Ethanol) Kit:**

Pattern agreement was assessed in pair-wise comparison between lots. There was 100% pattern agreement between the lots with manual and digital image reading.

Pattern agreement for NOVA View® Automated Fluorescence Microscopes ofware interpretation ranged from 71.9% to 75.8%. This was acceptable.

**Pattern agreement, NOVA Lite DAPI ANCA (Formalin) Kit:**

Pattern agreement was assessed in pair-wise comparison between lots. Pattern agreement between the lots with manual and digital image reading ranged from 90.9% to 100%. Pattern agreement for NOVA View® Automated Fluorescence Microscope software interpretation ranged from 81.8% to 87.9% (this includes positive/negative discrepancies, as well as discrepancy originating from NOVA View® Automated Fluorescence Microscope reporting Unrecognized pattern). This was acceptable.

## b. Linearity/assay reportable range:

Not applicable

## c. Traceability, Stability, Expected values (controls, calibrators, or methods):

### Traceability:

A recognized standard for anti-neutrophil cytoplasmic antibodies is not available.

### Stability:

A real-time stability study of the NOVA Lite DAPI ANCA (Ethanol) Kit with DAPI was conducted using two controls (pANCA Control and cANCA Control) that were prepared in a 2-fold serial dilution, five patient samples that were diluted using a 1:20 dilution, and undiluted controls [Negative, cANCA Positive, pANCA Positive, ANA Homogeneous, dsDNA Positive, Speckled Pattern and atypical ANCA (aANCA) Positive]. A control set (months 0) and a test set of slides (months 19) were run on NOVA View® Automated Fluorescence Microscope and manual and digital image interpretation was done. Testing was done 19 months after manufacturing date (1 month after claimed shelf life) to confirm 18-month expiration date.

To assess the real time stability of the NOVA Lite DAPI ANCA (Formalin) Kit with DAPI. The study was conducted using two controls, pANCA Control and cANCA Control that were prepared in a 2-fold serial dilution, seven patients that were diluted using a 1:20 dilution, and undiluted controls [Negative, cANCA Positive, pANCA Positive, ANA Homogeneous and aANCA Positive]. A control set and a test set of

16

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slides were run on NOVA View® Automated Fluorescence Microscope and manual and digital image interpretation was done. Testing was done 19 months after manufacturing date (1 month after claimed shelf life) to confirm 18-month expiration date.

Acceptance criteria were established to demonstrate consistency in fluorescence intensity of staining and patterns of fluorescence over the testing period. This study demonstrated that the reagents and substrate are stable out to at least 19 months, thereby validating the 19-month stability claim.

d. Detection limit:

Not applicable

e. Analytical specificity:

Interference:

The interference study was performed according to CLSI EP07-A2, Interference Testing in Clinical Chemistry; Approved Guideline - Second Edition. Interference by bilirubin, hemoglobin, triglycerides, cholesterol and rheumatoid factor (RF) IgM was assessed using the following final three testing concentrations:

| Interfering substance | Maximum final Concentration tested | Medium final Concentration tested | Minimum final concentration tested |
| --- | --- | --- | --- |
| Bilirubin, conjugated | 100 mg/dL | 50 mg/dL | 25 mg/dL |
| Hemoglobin | 200 mg/dL | 100 mg/dL | 50 mg/dL |
| Triglycerides | 1000 mg/dL | 500 mg/dL | 250 mg/dL |
| Cholesterol | 224.3 mg/dL | 112.1 mg/dL | 56 mg/mL |
| RF IgM | 56 IU/mL | 39 IU/mL | 28 IU/mL |
| Human IgG | 70 mg/mL | 35 mg/mL/mL | 17.5 mg/mL |
| Rituximab | 7.6 mg/mL | 3.8 mg/mL | 1.9 mg/mL |
| Methylprednisolone | 0.85 mg/mL | 0.42 mg/mL | 0.21 mg/mL |
| Cyclophosphamide | 4.1 mg/mL | 2.05 mg/mL | 1.03 mg/mL |
| Methotrexate | 0.01 mg/mL | 0.005 mg/mL | 0.0025 mg/mL |
| Azathioprine | 0.03 mg/mL | 0.015 mg/mL | 0.0075 mg/mL |

Five specimens were tested (one low anti-MPO positive, one strong anti-MPO positive, one low anti-PR3 positive, one strong anti-PR3 positive and one negative) in triplicate for each level of interferent. Interfering substances were spiked into every specimen at three different concentrations in $10\%$ of total specimen volume resulting in the final interferant concentrations indicated in the table above. To assess interference with RF, $10\%$ , $30\%$ and $50\%$ RF positive sample by volume was added to the test samples. Appropriate controls were made by adding $10\%$ sample diluent to the same samples. All samples were processed with NOVA Lite® DAPI ANA kit,

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and read with NOVA View® Automated Fluorescence Microscope. Digital images were interpreted and confirmed. All slides were also read by the same operator with manual microscopy.

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 the tested substances, up to the maximal concentrations indicated above.

## Cross-reactivity:

Cross reactivity with autoimmune and infectious agent antibodies was examined in the clinical patient population shown in the tables below (n=151), and 25%, 26% and 25% cross-reactivity was shown on Ethanol-fixed slides with NOVA View® Automated Fluorescence Microscope software interpretation, manual interpretation and digital image interpretation, and 8%, 8% and 11% cross-reactivity was shown on Formalin-fixed slides, respectively:

NOVA Lite DAPI ANCA (Ethanol) Kitcross-reactivity with autoimmune and infectious agent antibodies:

| Diagnosis | N | NOVA View® Automated Fluorescence Microscope positive | | Digital positive | | Manual positive | |
| --- | --- | --- | --- | --- | --- | --- | --- |
| Infectious Disease | 40 | 14 | 35% | 14 | 35% | 14 | 35% |
| Autoimmune thyroid disease | 15 | 14 | 93% | 13 | 87% | 13 | 87% |
| Celiac (tTG IgA POSITIVE) | 61 | 8 | 13% | 8 | 13% | 9 | 15% |
| Rheumatoid Arthritis (RA) | 35 | 2 | 6% | 2 | 6% | 3 | 9% |
| Total | 151 | 38 | 25% | 37 | 25% | 39 | 26% |

NOVA Lite DAPI ANCA (Formalin) Kit cross-reactivity with autoimmune and infectious agent antibodies:

| Diagnosis | N | NOVA View® Automated Fluorescence Microscope positive | | Digital positive | | Manual positive | |
| --- | --- | --- | --- | --- | --- | --- | --- |
| Infectious Disease | 40 | 3 | 8% | 3 | 8% | 2 | 5% |
| Autoimmune thyroid disease | 15 | 6 | 40% | 7 | 47% | 6 | 40% |
| Celiac (tTG IgA POSITIVE) | 61 | 3 | 5% | 4 | 7% | 3 | 5% |

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It is known that ANA and other anti-cell antibodies may interfere with ANCA detection by causing nuclear and/or cytoplasmic staining. It is assumed that the main cause of interference shown in the population above is the result of ANA in the samples.

Cross-reactivity caused by known ANA positive samples was examined by testing 25 analytically characterized samples, and cross reactivity was found with 76%, 80% and 80% of ANA positive samples on Ethanol-fixed slides with NOVA View® Automated Fluorescence Microscope software interpretation, manual interpretation and digital image interpretation, and 24%, 32% and 24% cross-reactivity was shown on Formalin-fixed slides, respectively:

NOVA Lite DAPI ANCA (Ethanol) Kitcross-reactivity with ANA:

| ANA positive sample | N | Number and % of Ethanol ANCA positive samples | | | | | |
| --- | --- | --- | --- | --- | --- | --- | --- |
| | | NOVA View positive | | Digital positive | | Manual positive | |
| Homogeneous | 5 | 5 | 100% | 5 | 100% | 5 | 100% |
| Speckled | 5 | 4 | 80% | 4 | 80% | 4 | 80% |
| Centromere | 5 | 5 | 100% | 5 | 100% | 5 | 100% |
| Nucleolar | 5 | 3 | 60% | 3 | 60% | 3 | 60% |
| Nuclear dots | 5 | 2 | 40% | 3 | 60% | 3 | 60% |
| Total | 25 | 19 | 76% | 20 | 80% | 20 | 80% |

NOVA Lite DAPI ANCA (Formalin) Kit cross-reactivity with ANA:

| ANA positive sample | N | Number and % of Formalin ANCA positive samples | | | | | |
| --- | --- | --- | --- | --- | --- | --- | --- |
| | | NOVA View® Automated Fluorescence Microscope positive | | Digital positive | | Manual positive | |
| Homogeneous | 5 | 4 | 80% | 4 | 80% | 4 | 80% |
| Speckled | 5 | 1 | 20% | 1 | 20% | 1 | 20% |
| Centromere | 5 | 1 | 20% | 1 | 20% | 1 | 20% |
| Nucleolar | 5 | 0 | 0% | 0 | 0% | 1 | 20% |
| Nuclear dots | 5 | 0 | 0% | 0 | 0% | 1 | 20% |
| Total | 25 | 6 | 24% | 6 | 24% | 8 | 32% |

The following statements were added to the package insert of the kits under "Limitations of the Procedure":

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NOVA Lite DAPI ANCA (Ethanol) Kit:

**Positive IIF results should be confirmed by antigen (MPO and PR3)-specific solid phase assays.**

ANA positive samples may react with the nuclei of ethanol-fixed neutrophils, masking or mimicking ANCA.

Any report of positive neutrophil fluorescence issued before the ELISA results are available should indicate that positive fluorescence alone is not specific for the diagnosis of granulomatosis with polyangiitis (GPA) or microscopic polyangiitis (MPA).

Decision about treatment should not be based solely on the ANCA results

NOVA Lite DAPI ANCA (Formalin) Kit:

Positive IIF results should be confirmed by antigen (MPO and PR3)-specific solid phase assays.

High-titer ANCA is suggestive of ANCA-associated vasculitis, but should not be considered diagnostic by itself. The ANCA result should be considered in combination with other serological results as well as the overall clinical history including signs and symptoms of the patient.

ANCA Formalin is not intended to be used by itself, but together with ANCA Ethanol test.

ANA positive samples may show weak nuclear fluorescence on formalin-fixed neutrophil granulocytes.

ANA positive samples may show weak nuclear fluorescence on formalin fixed neutrophil granulocytes.

Any report of positive neutrophil fluorescence issued before the ELISA results are available should indicate that positive fluorescence alone is not specific for the diagnosis of granulomatosis with polyangiitis (GPA) or microscopic polyangiitis (MPA).

Decisions about treatment should not be based solely on the ANCA results.

f. Assay cut-off:

20

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The assay cutoffs were established by testing 110 reference samples (n=90 healthy subjects and n=20 samples from patients with infections) at a 1:20 dilution. These were read by NOVA View® Automated Fluorescence Microscope and manually. Manual results were used as the reference. Based on this study the established cutoffs are 20 LIU for NOVA Lite DAPI ANCA (Ethanol) Kit and 30 LIU for NOVA Lite DAPI ANCA (Formalin) Kit. The above cut-off values were verified on an independent cohort, consisting of 89 samples from apparently healthy individuals. On this cohort, there were 4 positive results with NOVA View® Automated Fluorescence Microscopes software interpretation, eight with manual interpretation, and four with digital interpretation with NOVA Lite DAPI ANCA (Ethanol) Kit, and no positive results with any interpretation method with NOVA Lite DAPI ANCA (Formalin) Kit.

# 2. Comparison studies:

# a. Method comparison with predicate device:

The method comparison study included 267 samples comprised of MPO/PR3 positive specimens and disease control groups. Qualitative agreement, pattern agreement and grade agreement were calculated between NOVA View® Automated Fluorescence Microscope software interpretation, manual reading and digital image reading, each comparing the candidate device to the predicate device, at one internal site.

| Sample Source | n |
| --- | --- |
| Characterized MPO/PR3 | 119 |
| Infectious Disease | 20 |
| Systemic Lupus Erythematosus | 35 |
| Progressive Systemic Sclerosis | 20 |
| Rheumatoid arthritis | 34 |
| Inflammatory Bowel Disease | 21 |
| Chronic Kidney Disease | 18 |
| Total | 267 |

Comparison to the predicate: NOVA Lite DAPI ANCA (Ethanol) Kit Qualitative Agreement

| | Positive Agreement % (95% CI) | Negative Agreement % (95% CI) | Total Agreement % (95% CI) |
| --- | --- | --- | --- |
| Predicate Manual vs Candidate Manual | 91.8 (87.6–94.7) | 91.2 (77.0–97.0) | 91.8 (87.8–94.5) |
| Predicate Manual vs Candidate Digital | 80.3 (74.7–84.9) | 97.1 (85.1–99.5) | 82.4 (77.4–86.5) |
| Predicate Manual vs Candidate NOVA View® Automated Fluorescence Microscope | 76.4 (70.5–81.4) | 97.1 (85.1–99.5) | 79.0 (73.7–83.5) |

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# Grade Agreement:

Comparison of the grade assignments for the candidate device to that of the predicate device for both manual and digital reads demonstrated that of the 267 specimens tested, all but 1 (99.6%) yielded results that were within $\pm 2$ grades and $91\%$ were within $\pm 1$ grade.

# Pattern Agreement:

Comparison of the grade assignments for the candidate device to that of the predicate device for both manual and digital reads demonstrated that of the 267 specimens tested, $89.9\%$ of the manual reads of the candidate device and $80.1\%$ of the digital reads of the candidate device yielded pattern agreement with the predicate device (manually read).

Comparison to the predicate: NOVA Lite DAPI ANCA (Formalin) Kit Qualitative Agreement

| | Positive Agreement % (95% CI) | Negative Agreement % (95% CI) | Total Agreement % (95% CI) |
| --- | --- | --- | --- |
| Predicate Manual vs Candidate Manual | 94.4 (89.8–97.0) | 90.5 (83.4–94.7) | 92.9 (89.2–95.4) |
| Predicate Manual vs Candidate Digital | 79.0 (72.1–84.6) | 99.0 (94.8–99.8) | 86.9 (82.3–90.4) |
| Predicate Manual vs Candidate NOVA View® Automated Fluorescence Microscope | 72.2 (64.9–78.5) | 97.1 (91.9–99.0) | 82.0 (77.0–86.2) |

# Grade Agreement:

Comparison of the grade assignments for the candidate device to that of the predicate device for both manual and digital reads demonstrated that of the 267 specimens tested, all yielded results that were within $\pm 2$ grades and $99\%$ were within 1 grade.

# Pattern Agreement:

Comparison of the grade assignments for the candidate device to that of the predicate device for both manual and digital reads demonstrated that of the 267 specimens tested, $92.1\%$ of the manual reads of the candidate device and $86.9\%$ of the digital reads of the candidate device yielded pattern agreement with the predicate device (manually read).

# b. Device performance based on comparison to clinical diagnosis:

Prevalence of ANCA positivity on Formalin and Ethanol slides for NOVA View®

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Automated Fluorescence Microscopemanual reading and digital interpretation was established for each diagnostic group tested. The ANCA associated vasculitis (AAV) classification group typically includes granulomatosis with polyangiitis (GPA) (formerly Wegener's granulomatosis), microscopic polyangiitis (MPA), and eosinophilic granulomatosis with polyangiitis (eGPA) (formerly Churg-Strauss syndrome). Where the specific diagnosis was identified, results were presented for each condition as well as for analyses for which the AAV diagnostic subsets (GPA, eGPA or MPA) were not specified.

Performance by disease status:

| Disease status | # of samples | NOVA View® Automated Fluorescence Microscope (% Positive) | | Manual (% Positive) | | Digital (% Positive) | |
| --- | --- | --- | --- | --- | --- | --- | --- |
| | | Ethanol | Formalin | Ethanol | Formalin | Ethanol | Formalin |
| ANCA Associated Vasculitis (AAV) | 185 | 60 | 61 | 71 | 75 | 70 | 72 |
| Characterized MPO/PR3 | 59 | 97 | 95 | 100 | 100 | 100 | 98 |
| Infectious Disease | 40* | 13 | 10 | 30 | 15 | 23 | 13 |
| Autoimmune thyroid disease | 15* | 93 | 40 | 87 | 40 | 87 | 47 |
| Chronic Kidney Disease (CKD) | 18* | 50 | 0 | 72 | 6 | 56 | 6 |
| Progressive Systemic Sclerosis | 25* | 56 | 4 | 72 | 12 | 56 | 8 |
| Systemic Lupus Erythematosus | 23 | 70 | 17 | 78 | 39 | 65 | 22 |
| Dermatitis | 12* | 25 | 0 | 33 | 0 | 25 | 0 |
| Diabetes Type II | 20 | 40 | 0 | 40 | 0 | 40 | 5 |
| Celiac (tTG IgA POSITIVE) | 61 | 18 | 8 | 23 | 7 | 21 | 5 |
| Chronic Obstructive Pulmonary Disease (COPD) | 20 | 40 | 0 | 45 | 10 | 45 | 10 |
| Rheumatoid Arthritis (RA) | 35* | 71 | 17 | 74 | 14 | 71 | 14 |
| Crohn's Disease | 11* | 55 | 27 | 55 | 18 | 55 | 18 |

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| Disease status | # of samples | NOVA View® Automated Fluorescence Microscope (% Positive) | | Manual (% Positive) | | Digital (% Positive) | |
| --- | --- | --- | --- | --- | --- | --- | --- |
| | | Ethanol | Formalin | Ethanol | Formalin | Ethanol | Formalin |
| Ulcerative Colitis | 15* | 27 | 0 | 53 | 13 | 33 | 7 |
| Sinusitis | 18* | 6 | 0 | 28 | 6 | 11 | 0 |
| Allergy | 7* | 29 | 0 | 43 | 14 | 29 | 29 |
| Healthy controls | 89 | 4 | 0 | 9 | 0 | 4 | 0 |
| Total | 653 | | | | | | |

*used for specificity calculation

Performance on AAV samples by diagnosis:

| Diagnosis | # of samples | NOVA View® Automated Fluorescence Microscope (% Positive) | | Manual (% Positive) | | Digital (% Positive) | |
| --- | --- | --- | --- | --- | --- | --- | --- |
| | | Ethanol | Formalin | Ethanol | Formalin | Ethanol | Formalin |
| GPA | 113 | 47 | 59 | 59 | 73 | 57 | 71 |
| MPA | 21 | 86 | 57 | 95 | 71 | 95 | 71 |
| eGPA | 12 | 25 | 17 | 50 | 25 | 42 | 25 |
| Undifferentiated AAV | 39 | 95 | 82 | 97 | 97 | 100 | 93 |
| Total | 185 | | | | | | |

Conjugate comparison:

The NOVA Lite® DAPI ANCA Ethanol and Formalin Kits contain the same conjugate as that of the predicate device, but with blue fluorescent dye DAPI (4',6-diamidino-2-phenylindole) added. To adapt the assay for use on NOVA View® Automated Fluorescence Microscope, the blue fluorescent dye DAPI (4',6-diamidino-2-phenylindole) that binds strongly to A-T rich regions in DNA, was added to the conjugate.

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

Thirty-six individual serum samples have been tested (analytically characterized serum samples and controls), in addition to a diluent blank, with FITC IgG Conjugate (508113) and FITC IgG Conjugate with DAPI (508102), using NOVA Lite DAPI ANCA (Ethanol) Kit and NOVA Lite DAPI ANCA (Formalin) Kit reagents. Two sets of slides were stained: one with the conjugate without DAPI (the predicate

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device), the other with the conjugate using NOVA Lite DAPI ANCA (Ethanol) Kit and NOVA Lite DAPI ANCA (Formalin) Kit reagents. Results were manually interpreted.

| Ethanol | Conjugate with DAPI (Grade) | | | | | |
| --- | --- | --- | --- | --- | --- | --- |
| Without DAPI – (Grade) | 0 | 2 | 3 | | 4 | Total |
| 0 | 14 | 0 | 0 | | 0 | 14 |
| 2 | 0 | 2 | 1 | | 0 | 3 |
| 3 | 0 | 0 | 5 | | 2 | 7 |
| 4 | 0 | 0 | 1 | | 11 | 12 |
| Total | 14 | 2 | 7 | | 13 | 36 |
| Formalin | Conjugate with DAPI (Grade) | | | | | |
| Without DAPI – (Grade) | 0 | 1 | 2 | 3 | 4 | Total |
| 0 | 20 | 0 | 0 | 0 | 0 | 20 |
| 2 | 0 | 2 | 0 | 0 | 0 | 2 |
| 3 | 0 | 0 | 1 | 1 | 0 | 2 |
| 4 | 0 | 0 | 0 | 1 | 0 | 1 |
| Total | 20 | 2 | 1 | 3 | 10 | 36 |

Endpoint titers obtained with the new conjugate were within $\pm 1$ dilution step of those obtained with the predicate conjugate. Results are presented in the table below:

| | Endpoint titer, Ethanol | | Endpoint titer, Formalin | |
| --- | --- | --- | --- | --- |
| | w/o DAPI | w/ DAPI | w/o DAPI | w/ DAPI |
| pANCA positive | 320 | 320 | 320 | 320 |
| pANCA positive | 640 | 640 | 320 | 320 |
| pANCA positive | 1280 | 1280 | 320 | 320 |
| cANCA positive | 5120 | 5120 | 2560 | 2560 |
| cANCA positive | 1280 | 2560 | 1280 | 1280 |
| cANCA positive | 1280 | 1280 | 2560 | 2560 |

# b. Performance on Standardized Reference Materials

The US Centers for Disease Control and Prevention / International Union of Immunological Societies reference materials for MPO and PR3 and the European reference material for MPO (DA476/IFCC) were tested and the results were consistent with the expected results and were within $+/- 2$ dilution steps among the different methods. These results for endpoint titrations, are summarized below for

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ethanol and formalin fixed slides.

| | | Titration | | | |
| --- | --- | --- | --- | --- | --- |
| NOVA Lite DAPI ANCA (Ethanol) Kit | Published Value | NOVA View® Automated Fluorescence Microscope | Single Well Titer (SWT) | Manual | Digital |
| CDC/IUIS Sample 15 - L/N: 07-0001 | 100 IU/mL MPO ANCA | 160 | 160 | 320 | 320 |
| CDC/IUIS Sample 16 - L/N: 07-0002 | 100 IU/mL PR3 ANCA | 40 | 80 | 80 | 160 |
| International Sample 1 ERM-DA476/IFCC L/N:0091 | 84 mg/L | 320 | 1280 | 1280 | 640 |
| | | Titration | | | |
| --- | --- | --- | --- | --- | --- |
| NOVA Lite DAPI ANCA (Formalin) Kit | Published Value | NOVA View® Automated Fluorescence Microscope | SWT | Manual | Digital |
| CDC Sample 15 - L/N: 07-0001 | 100 IU/mL MPO ANCA | 80 | NA | 160 | 160 |
| CDC Sample 16 - L/N: 07-0002 | 100 IU/mL PR3 ANCA | 80 | NA | 320 | 160 |
| International Sample 1 ERM-DA476/IFCC L/N:0091 | 84 mg/L | 160 | NA | 640 | 320 |

c. Matrix comparison:

Not applicable

# 3. Clinical studies:

a. Clinical Sensitivity and Specificity:

To assess clinical performance, a clinical study was performed on 653 clinically or analytically characterized serum samples. Fifty nine samples were analytically characterized (anti-MPO/PR3 positive), and 89 were healthy controls (these samples were excluded from the sensitivity and specificity calculations).

Additionally, 287 clinically or analytically characterized serum samples were tested at a sponsor lab (Site#1) and at two external clinical sites (Site#2 and Site #3) with both Ethanol and Formalin ANCA. The cohort contained 49 analytically characterized samples that were excluded from the sensitivity and specificity

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calculations (clinical cohort $n = 238$ ).

The composition of the combined clinical cohort $(n = 653)$ is described in the "Method comparison" section.

Patients were diagnosed according to current classification criteria $^{1,2,3}$ and ANCA testing. Sensitivity (on AAV) and specificity, calculated on the combined population, are shown below. The AAV classification group typically includesGPA, MPA, andeGPA. Where the specific diagnosis was identified, results were presented for each condition separately as well as for the specimens for which the AAV diagnostic subsets (GPA, eGPA or MPA) were not specified

NOVA Lite DAPI ANCA (Ethanol) Kit Digital:

| | cANCA Pattern | | pANCA Pattern | |
| --- | --- | --- | --- | --- |
| | Sensitivity (%) (95% CI) | Specificity (%) (95% CI) | Sensitivity (%) (95% CI) | Specificity (%) (95% CI) |
| GPA (n=113) | 29.2 (21.6–38.2) | 91.6 (88.0–94.1) | 27.4 (20.1–36.3) | 66.6 (61.2–71.5) |
| MPA (n=21) | 4.8 (0.8–22.7) | 91.6 (88.0–94.1) | 90.5 (71.1–97.3) | 66.6 (61.2–71.5) |
| eGPA (n=12) | 8.3 (1.5–35.4) | 91.6 (88.0–94.1) | 33.3 (13.8–60.9) | 66.6 (61.2–71.5) |
| AAV (n=39) | 24.0 (17.8–31.5) | 91.6 (88.0–94.1) | 37.0 (29.6–45.1) | 66.6 (61.2–71.5) |

NOVA Lite DAPI ANCA (Ethanol) Kit Manual

| | cANCA Pattern | | pANCA Pattern | |
| --- | --- | --- | --- | --- |
| | Sensitivity (%) (95% CI) | Specificity (%) (95% CI) | Sensitivity (%) (95% CI) | Specificity (%) (95% CI) |
| GPA (n=113) | 29.2 (21.6–38.2) | 91.3 (87.6–93.9) | 30.1 (22.4–39.1) | 59.7 (54.2–64.9) |
| MPA (n=21) | 4.8 (0.8–22.7) | 91.3 (87.6–93.9) | 90.5 (71.1–97.3) | 59.7 (54.2–64.9) |
| eGPA (n=12) | 8.3 (1.5–35.4) | 91.3 (87.6–93.9) | 33.3 (13.8–60.9) | 59.7 (54.2–64.9) |
| AAV (n=39) | 24.0 (17.8–31.5) | 91.3 (87.6–93.9) | 39.0 (31.5–47.1) | 59.7 (54.2–64.9) |

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NOVA Lite DAPI ANCA (Ethanol) Kit:

| | cANCA Pattern | | pANCA Pattern | |
| --- | --- | --- | --- | --- |
| | Sensitivity (%) (95% CI) | Specificity (%) (95% CI) | Sensitivity (%) (95% CI) | Specificity (%) (95% CI) |
| GPA (n=113) | 12.4 (7.2–20.4) | 95.9 (93.0–97.7) | 30.1 (22.4–39.1) | 69.6 (64.1–74.6) |
| MPA (n=21) | 0.0 (0.0–15.5) | 95.9 (93.0–97.7) | 85.7 (65.4–95.0) | 69.6 (64.1–74.6) |
| eGPA (n=12) | 0.0 (0.0–25.9) | 95.9 (93.0–97.7) | 18.2 (5.1–47.7) | 69.6 (64.1–74.6) |
| AAV (n=39) | 9.3 (5.4–15.6) | 95.9 (93.0–97.7) | 9.3 (5.4–15.6) | 69.6 (64.1–74.6) |

NOVA Lite DAPI ANCA (Formalin) Kit Digital:

| | cANCA Pattern | |
| --- | --- | --- |
| | Sensitivity (%) (95% CI) | Specificity (%) (95% CI) |
| GPA | 70.8 (61.8–78.4) | 91.3 (87.6–93.9) |
| MPA | 71.4 (50.0–86.2) | 91.3 (87.6–93.9) |
| eGPA | 25.0 (8.9–53.2) | 91.3 (87.6–93.9) |
| AAV | 67.1 (59.1–74.2) | 91.3 (87.6–93.9) |

NOVA Lite DAPI ANCA (Formalin) Kit Manual:

| | cANCA Pattern | |
| --- | --- | --- |
| | Sensitivity (%) (95% CI) | Specificity (%) (95% CI) |
| GPA | 73.5 (64.6–80.7) | 89.7 (85.9–92.6) |
| MPA | 71.4 (50.0–86.2) | 89.7 (85.9–92.6) |
| eGPA | 16.7 (4.7–44.8) | 89.7 (85.9–92.6) |
| AAV | 68.5 (60.6–75.5) | 89.7 (85.9–92.6) |

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Formalin NOVA View® Automated Fluorescence Microscope:

| | cANCA Pattern | |
| --- | --- | --- |
| | Sensitivity (%) (95% CI) | Specificity (%) (95% CI) |
| GPA | 38.7 (28.5–50.0) | 98.7 (96.6–99.5) |
| MPA | 47.1 (26.2–69.0) | 98.7 (96.6–99.5) |
| eGPA | 16.7 (4.7–44.8) | 98.7 (96.6–99.5) |
| AAV | 37.5 (28.8–47.1) | 98.7 (96.6–99.5) |

Sensitivity and specificity were also calculated on the 238 clinical samples that were run during the between-site reproducibility study at all three sites. The results are presented in the tables below:

Site 1:

NOVA Lite DAPI ANCA (Ethanol) Kit

| N=238 | Sensitivity % (95% CI) | Specificity % (95% CI) |
| --- | --- | --- |
| Manual Read | 88.8 (80.0–94.0) | 50.6 (42.9–58.3) |
| Digital Read | 87.5 (78.5–93.1) | 57.6 (49.8–65.0) |
| NOVA View® Automated Fluorescence Microscope | 85 (75.6–91.2) | 62.7 (54.9–69.8) |

NOVA Lite DAPI ANCA (Formalin) Kit

| N=238 | Sensitivity % (95% CI) | Specificity % (95% CI) |
| --- | --- | --- |
| Manual Read | 92.5 (84.6–96.5) | 90.5 (84.9–94.2) |
| Digital Read | 88.8 (80.0–94.0) | 92.4 (87.2–95.6) |
| NOVA View® Automated | 83.8 (74.2–90.3) | 96.8 (92.8–98.6) |

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| Fluorescence Microscope | | |
| --- | --- | --- |

Site 2:

NOVA Lite DAPI ANCA (Ethanol) Kit

| N=238 | Sensitivity % (95% CI) | Specificity % (95% CI) |
| --- | --- | --- |
| Manual Read | 75 (64.5–83.2) | 57.0 (49.2–64.4) |
| Digital Read | 71.3 (60.5–80.0) | 65.6 (57.9–72.6) |
| NOVA View® Automated Fluorescence Microscope | 70.0 (59.2–78.9) | 69.5 (61.8–76.2) |

NOVA Lite DAPI ANCA (Formalin) Kit

| N=238 | Sensitivity % (95% CI) | Specificity % (95% CI) |
| --- | --- | --- |
| Manual Read | 80.0 (70.0–87.3) | 90.5 (84.9–94.2) |
| Digital Read | 81.3 (71.3–88.3) | 94.3 (89.5–97.0) |
| NOVA View® Automated Fluorescence Microscope | 76.3 (65.9–84.2) | 94.3 (89.5–97.0) |

Site 3:

NOVA Lite DAPI ANCA (Ethanol) Kit

| N=238 | Sensitivity % (95% CI) | Specificity % (95% CI) |
| --- | --- | --- |
| Manual Read | 72.5 (61.9–81.1) | 63.9 (56.2–71.0) |
| Digital Read | 72.5 (61.9–81.1) | 65.8 (58.1–72.8) |
| NOVA View® Automated | 70.0 (59.2–78.9) | 68.4 (60.7–75.1) |

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31

| Fluorescence
Microscope | | |
| --- | --- | --- |

NOVA Lite DAPI ANCA (Formalin) Kit

| N=238 | Sensitivity %
(95% CI) | Specificity %
(95% CI) |
| --- | --- | --- |
| Manual Read | 77.5
(67.2–85.3) | 93.0
(88.0–96.1) |
| Digital Read | 76.3
(65.9–84.2) | 94.3
(89.5–97.0) |
| NOVA View® Automated Fluorescence Microscope | 75.0
(64.5–83.2) | 96.8
(92.8–98.6) |

b. Other clinical supportive data (when a. and b. are not applicable):

Not applicable

4. Clinical cut-off:

See analytical cut-off.

5. Expected values/Reference range:

Expected values were analyzed on 89 samples from apparently healthy subjects: 29 females, 54 males, 6 with unknown gender, with mean age of 45.7 years (range of 18-68). With NOVA Lite DAPI ANCA (Ethanol) Kit, there were four positive results with NOVA View® Automated Fluorescence Microscopes ofware interpretation, eight with manual interpretation, and four with digital interpretation.

There were no positive results with NOVA Lite DAPI ANCA (Formalin) Kit testing, by any interpretation method.

N. Proposed Labeling:

The labeling is sufficient and it satisfies the requirements of 21 CFR Part 809.10.

O. Conclusion:

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