NOVA Lite® DAPI ANCA (Ethanol) Kit is an indirect immunofluorescence assay for the qualitative detection and semiquantitative 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 istoypes 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 Description

The NOVA Lite DAPI ANCA (Ethanol) and 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:

. 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 PR3 and 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

AI/ML Overview

This document outlines the acceptance criteria and study results for the NOVA Lite Dapi ANCA Ethanol Kit and Formalin Kit. This is a medical device, and the information is presented in the context of an FDA 510(k) premarket notification.

Acceptance Criteria and Reported Device Performance

The device is evaluated based on its precision performance (within laboratory imprecision, between lots reproducibility, between sites/instruments reproducibility, and between operators reproducibility), interference resistance, cross-reactivity with other conditions, and clinical sensitivity and specificity.

Precision Performance:

The acceptance criteria for precision studies consistently revolve around:

Qualitative agreement: ≥ 90% (for NOVA View, Digital, and Manual readings)
Grade agreement: ≥ 90% within ± 1 reactivity grade (for Digital and Manual readings)
Pattern agreement: ≥ 90% (for Digital and Manual readings), or ≥ 80% after excluding positive/negative discrepancies for NOVA View

The studies generally show the device meets these targets. For example:

Within-laboratory imprecision: "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."
Between lots reproducibility: All qualitative agreements for Ethanol ANCA (NOVA View, Manual, Digital) were ≥ 97.0%. For Formalin ANCA, agreements ranged from 90.9% to 100%. Grade agreements were 100% within ± 1 grade for both Ethanol and Formalin ANCA for manual and digital readings. Pattern agreements were 100% for manual and digital Ethanol ANCA, and ranged from 90.9% to 100% for manual and digital Formalin ANCA.
Between sites/instruments reproducibility:
- Ethanol ANCA: Qualitative agreement (Total) ranged from 90.9% to 96.1% for NOVA View, 86.4% to 91.5% for Manual, and 90.1% to 96.1% for Digital. Grade agreement was ≥ 96.0% for both Manual and Digital. Pattern agreement (excluding pos/neg disagreement) was ≥ 90.0% for NOVA View, ≥ 98.0% for Manual, and ≥ 98.0% for Digital.
- Formalin ANCA: Qualitative agreement (Total) ranged from 93.7% to 94.8% for NOVA View, 90.2% to 91.3% for Manual, and 92.7% to 95.5% for Digital. Grade agreement was ≥ 91.0% for both Manual and Digital. Pattern agreement (excluding pos/neg disagreement) was ≥ 93.0% for NOVA View, ≥ 98.0% for Manual, and ≥ 99.0% for Digital.
Between operators reproducibility: Overall agreement (Positive/Negative) for Ethanol ANCA ranged from 94.4% to 100% for Manual reading and 97.2% to 100% for Digital Image Reading. For Formalin ANCA, it ranged from 94.0% to 100.0% for Manual reading and 91.6% to 99.6% for Digital Image Reading.

Interference:

Acceptance Criteria: Grades obtained on samples with interfering substances are within ± 1 reactivity grade of those obtained on the control samples, spiked with diluent.
Reported Performance: "No interference was detected with bilirubin up to 100 mg/dL, hemoglobin up to 200 mg/dL, triglycerides up to 1000 mg/dL, cholesterol up to 224.3 mg/dL, RF IgM up to 56 IU/mL, Human Immunoglobulin up to 35 mg/dL, Rituximab up to 7.6 mg/mL, Methylprednisolone up to 0.85 mg/mL, Cyclophosphamide up to 4.1 mg/mL, Methotrexate up to 0.01 mg/mL and Azathioprine up to 0.03 mg/mL. Reactivity grades of samples containing the interfering substance were within ± one grade of the control samples with both manual and digital reading."

Cross-reactivity:

The document reports cross-reactivity rates for various autoimmune and infectious conditions (e.g., Infectious Disease, Autoimmune thyroid disease, Celiac, Rheumatoid Arthritis). It also specifically examines cross-reactivity with known ANA positive samples. This is presented as information rather than having explicit numerical acceptance criteria in the provided text. The document acknowledges that ANA can interfere and states: "ANA positive samples may react with the nuclei of ethanol-fixed neutrophils, masking or mimicking ANCA. Positive IIF results should be confirmed by antigen specific solid phase assay for anti-MPO and anti-PR3."

Conjugate Comparison (to predicate device):

Acceptance Criteria: Qualitative agreement: ≥ 90%, Grade agreement: ≥ 90% within ± 1 reactivity grade, Endpoint dilution is within ±1 dilution step between the two conjugates.
Reported Performance: "The qualitative agreement and the grade agreement between the result obtained with the predicate and the new conjugate was 100% on both Ethanol and Formalin ANCA slides." Endpoint titers were within ±1 dilution step.
Method Comparison (to predicate device):
- Acceptance Criteria: Qualitative agreement: ≥ 80% (for manual and digital), Grade agreement: ≥ 90% within ± 1 reactivity grade, Pattern agreement: ≥ 80% between manual and digital interpretation.
- Reported Performance: Ethanol ANCA qualitative agreement ranged from 80.3% to 91.8% for manual and digital vs. predicate manual. Grade agreement (within ±2 grades) was 99.6%. Pattern agreement was ≥ 80.1%. Formalin ANCA qualitative agreement ranged from 79.0% to 94.4%. Grade agreement (within ±2 grades) was 100%. Pattern agreement was ≥ 86.9%.

Clinical Sensitivity and Specificity:

These are reported for various ANCA Associated Vasculitides (AAV) subgroups (GPA, MPA, eGPA) and overall AAV, as well as for control populations across multiple sites and interpretation methods (Digital, Manual, NOVA View). No explicit numerical acceptance criteria are given for these performance characteristics in this document, but the results are presented as the device's performance.

1. Table of Acceptance Criteria and Reported Device Performance

Category	Acceptance Criteria	Reported Device Performance (Summary)
Precision
- Within-lab Imprecision	Diff. within run ± 1 reactivity grade; Avg. diff. between runs ± 1 reactivity grade.	Met: "grades were within ± one reactivity grade within one run... and the average grade was no more than one reactivity grade different between runs." (p. 11)
- Between-lots	Qualitative agreement ≥ 90%; Grade agreement ≥ 90% (within ± 1 grade); Pattern agreement ≥ 90%.	Met: Qualitative agreements ≥ 90.9% (NOVA View), 100% (Manual/Digital). Grade agreements 100% (within ± 1 grade). Pattern agreements ≥ 71.9% (NOVA View), 100% (Manual), ≥ 90.9% (Digital). (p. 16-20)
- Between Sites/Instruments	Qualitative agreement ≥ 85%; Grade agreement ≥ 90% (within ± 1 grade); Pattern agreement ≥ 80% (excl. pos/neg disc.).	Met: Qualitative agreements ≥ 86.4% (Manual), ≥ 90.1% (Digital), ≥ 90.9% (NOVA View). Grade agreements ≥ 91.0%. Pattern agreements ≥ 90.0% (NOVA View), ≥ 98.0% (Manual/Digital ethanol), ≥ 93.0% (NOVA View), 99.0%-100% (Manual/Digital formalin). (p. 24-27)
- Between Operators	(Implicitly part of between-sites/instruments, but separate summary provided)	Positive/Negative overall agreement 94.0-100% (Manual), 91.6-100% (Digital) for Ethanol and Formalin ANCA. (p. 30, 33)
Interference	Grades obtained on samples with interfering substances are within ± 1 reactivity grade of controls.	Met: "No interference was detected with bilirubin... hemoglobin... triglycerides... cholesterol... RF IgM... Human Immunoglobulin... Rituximab... Methy
Conjugate Comparison	Qualitative agreement ≥ 90%; Grade agreement ≥ 90% (within ± 1 grade); Endpoint within ±1 dilution step.	Met: Qualitative and Grade agreement 100%. Endpoint titers within ±1 dilution step. (p. 37)
Method Comparison	Qualitative agreement ≥ 80% (Manual/Digital); Grade agreement ≥ 90% (within ± 1 grade); Pattern agreement ≥ 80% (Manual/Digital).	Met: Ethanol ANCA Qualitative 80.3-91.8% (Manual/Digital). Grade agreement 99.6% (within ±2 grades), Pattern 80.1-89.9%. Formalin ANCA Qualitative 79.0-94.4%. Grade agreement 100% (within ±2 grades), Pattern 86.9-92.1%. (p. 38-40)
SWT Function	SWT is within ± 2 dilution steps of manual titer AND digital titer.	Met: 97.7% of SWT results were within ± 2 dilution steps of both the manual and digital titer (for in-house validation). 100% for external sites. (p. 47)

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

Precision (within lab): 16 samples (3 negative, 13 positive - 7 anti-MPO, 6 anti-PR3) tested in triplicates across 10 runs (30 data points per sample). (p. 11) Data provenance is in-house (Inova Diagnostics). This was a prospective study.
Precision (between lots): 33 clinically and/or analytically characterized samples. (p. 16) Data provenance is in-house (Inova Diagnostics). This was a prospective study.
Precision (between sites/instruments): 287 clinically characterized samples were tested at three sites (Inova's laboratory and two external US clinical laboratories). Additionally, the two external sites each tested 100 routine clinical samples. The internal study uses 287 samples, with a clinical cohort of n=238 after excluding 49 analytically characterized samples. (p. 21) Data provenance includes US clinical laboratories (prospective, as samples were "tested").
Precision (between operators): 10 samples (2 negative, 4 P-ANCA, 4 C-ANCA positive) tested at each of three sites, for 5 days in 5 replicates (25 data points per sample). (p. 28) Data provenance includes US clinical laboratories (prospective).
Interference: 5 specimens (1 negative, 1 low MPO, 1 strong MPO, 1 low PR3, 1 strong PR3). These were spiked with various interferents and tested in triplicates. (p. 34) Data provenance is likely in-house. This was a prospective study.
Cross-reactivity: 151 clinical patient samples (Infectious Disease, Autoimmune thyroid disease, Celiac, Rheumatoid Arthritis) and 25 analytically characterized ANA positive samples. (p. 35) Data provenance includes a clinical patient population. This was likely a retrospective analysis from collected samples.
Conjugate Comparison: 36 specimens (analytically characterized serum samples and controls) plus a diluent blank. Endpoint titration on 6 positive samples. (p. 37) Data provenance is likely in-house. This was a prospective study.
Method Comparison (vs. predicate): 100 samples (50 P-ANCA, 50 C-ANCA) and various disease control groups (Infectious Disease, Systemic Lupus Erythematosus, Progressive Systemic Sclerosis, Rheumatoid arthritis and Chronic Kidney Disease) for a total of 267 samples. (p. 38) Data provenance is in-house (Inova Diagnostics), likely retrospective from collected samples.
Clinical Performance (Sensitivity/Specificity): 653 clinically or analytically characterized serum samples. A subset of 287 samples was also tested across three sites, with a clinical cohort of 238 after excluding analytically characterized samples. (p. 41) Data provenance covers a combined population, including US clinical samples and characterized samples. Likely a mix of retrospective and prospective.
Expected Values: 89 samples from apparently healthy subjects. (p. 45) Data provenance is not explicitly stated as US or international but is part of the broader clinical validation. Likely a retrospective analysis.

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

The document does not explicitly state the number of experts or their specific qualifications (e.g., "radiologist with 10 years of experience") used to establish the ground truth for the test sets.

For "clinically characterized samples" and "analytically characterized MPO/PR3" samples, it is implied that a reference standard (e.g., diagnosis of ANCA Associated Vasculitis, characterized anti-MPO/PR3 status) was used as ground truth. However, the exact process of how this ground truth was established, who established it, and their qualifications are not detailed.

The manual readings and digital image interpretations are performed by "trained operators."

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

The document does not describe an explicit adjudication method like "2+1" or "3+1" for discrepancies in the test sets.

For manual and digital readings in precision and method comparison studies, results from different operators/sites are compared.
The "NOVA View interpretation" results are expected to be reviewed and confirmed by a "trained operator." (p. 9, 45) This implies a human-in-the-loop confirmation process as a form of adjudication for the automated results.

5. If a multi-reader multi-case (MRMC) comparative effectiveness study was done, If so, what was the effect size of how much human readers improve with AI vs without AI assistance

While there are studies involving multiple sites and operators comparing manual reading, digital image reading (human reading of images captured by the automated system), and NOVA View software interpretation, the document does not describe a formal MRMC comparative effectiveness study in the sense of measuring the improvement of human readers with AI assistance vs. without AI assistance.

The studies compare the performance of human readers (manual and digital) and the NOVA View software alone, but not the synergistic effect or comparative effectiveness of AI-assisted human reading against unassisted human reading. The "digital image reading" is human reading of images captured by the NOVA View, which is an output of the system, but the document does not present data on how this assistance (providing the images) improves human readers compared to traditional manual microscopy.

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

Yes, a standalone performance (algorithm only) was done. The "NOVA View software interpretation" is explicitly compared throughout the document to "Manual reading" (traditional microscopy) and "Digital reading" (human interpretation of the NOVA View generated digital images).

For example, in the Clinical Sensitivity and Specificity section (p. 42-44), separate results are provided for "NOVA View" (software interpretation), "Digital" (human interpretation of digital images), and "Manual" (traditional microscopy). This clearly indicates a standalone performance evaluation of the NOVA View software. The statement "A trained operator must confirm results when generated with the NOVA View device" (p. 2-3, 45) specifies the intended use model, but the software's performance without this confirmation step is also reported.

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

The types of ground truth used include:

Analytically characterized samples: These are identified as "anti-MPO/PR3 positive" (p. 41), implying a biochemical or molecular characterization. This forms a strong ground truth for the presence of specific antibodies.
Clinically characterized serum samples: These are often categorized by "Diagnosis" (e.g., ANCA Associated Vaculitidies (AAV), Infectious Disease, Rheumatoid Arthritis, etc.) (p. 21-23, 41). The establishment of these diagnoses would likely be based on a combination of clinical findings, laboratory tests, histology, and possibly expert consensus from treating physicians. The document does not specify the exact diagnostic criteria or who established these clinical diagnoses.
Apparently healthy subjects: Used as a negative control group. (p. 41, 45)

8. The sample size for the training set

The document does not explicitly state the sample size for a "training set" for the NOVA View AI algorithm itself. It mentions that the "SWT function was established on 10 anti-MPO (P-ANCA) and 10 anti-PR3 (C-ANCA) positive samples" to establish LIU curves, which could be considered a form of training or calibration data for that specific function. However, a general training set size for the core ANCA detection and pattern recognition algorithm is not provided.

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

As the document does not explicitly detail a separate "training set" and its ground truth establishment, the information is limited. For the 20 samples (10 anti-MPO, 10 anti-PR3) used to establish the SWT function's LIU curves, the ground truth was "manually titrated" and "results were interpreted by NOVA View and by manual reading." (p. 47) This implies that the manual titration and reading served as the reference for establishing the LIU curves.

Summary

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

February 3, 2017

INOVA DIAGNOSTICS, INC. MICHAEL MAHLER VICE PRESIDENT OF ASSAY DEVELOPMENT 9900 OLD GROVE ROAD SAN DIEGO CA 92131

Re: K161258

Trade/Device Name: Nova Lite Dapi ANCA Ethanol Kit, Nova Lite Dapi ANCA Formalin Kit

Regulation Number: 21 CFR 866.5100 Regulation Name: Antinuclear antibody immunological test system Regulatory Class: II Product Code: MOB Dated: December 30, 2016 Received: January 3, 2017

Dear Dr. Mahler:

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 (OS) regulation (21 CFR Part 820); and if applicable, the electronic product radiation control provisions (Sections 531-542 of the Act); 21 CFR 1000-1050.

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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 (800) 638 2041 or (301) 796-7100 or at its Internet address

http://www.fda.gov/MedicalDevices/ResourcesforYou/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 (800) 638-2041 or (301) 796-7100 or at its Internet address

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

Sincerely yours,

Leonthena R. Carrington -S

Leonthena 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)

Device Name

NOVA Lite DAPI ANCA (Ethanol) NOVA Lite DAPI ANCA (Formalin)

Indications for Use (Describe)

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

Prescription Use (Part 21 CFR 801 Subpart D)
Over-The-Counter Use (21 CFR 801 Subpart C)

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NOVA Lite® DAPI ANCA (Ethanol) Kit, NOVA Lite® DAPI ANCA (Formalin) 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.

1.		Sponsor
2.		Purpose of submission
3.		Devices in the submission
4.		Primary contact
5.		Secondary contact
6.		Product Information and Classification
6.1		Proprietary and established names
6.2		Regulatory information
7.		Intended Use
8.		Indications for Use
9.		Predicate device
10.		Comparison matrix to predicate device
11.		Type of the product
12.		Device description
13.		Principle of the method and summary of the procedure
14.		Analytical performance characteristics
	14.1	Nomenclature used in the studies
	14.2	Precision performance
	14.2.1	Within laboratory imprecision
	14.2.2	Between lots reproducibility
	14.2.3	Between sites/instruments reproducibility
	14.2.4	Between operators reproducibility
	14.3	Linearity and Analytical Measuring Range (AMR)
	14.4	Interference
14.5	Cross-reactivity	32
15.	Cutoff	34
16.	Comparison with the predicate device	34
16.1	Conjugate comparison	34
16.2	Method comparison	35
17.	Clinical performance	38
17.1	Clinical sensitivity and specificity	39
17.2	Expected values	43
18.	System description	43
18.1	NOVA View Automated Fluorescence Microscope and Software	43
18.1.1	NOVA View Automated Fluorescence Microscope	43
18.1.2	Software	43

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1. Sponsor

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 ANCA (Ethanol) Kit NOVA Lite® DAPI ANCA (Formalin) Kit

4. Primary contact

Michael Mahler, VP of Research and Development Inova Diagnostics, Inc 9900 Old Grove Road, San Diego, CA, 92131 Phone: 858-586-9900/1356 email: mmahler@inovadx.com

5. Secondary contact

Ronda Elliott, VP of Quality Systems and Regulatory Affairs Inova Diagnostics, Inc 9900 Old Grove Road, San Diego, CA, 92131 Phone: 858-586-9900/1381 Fax: 858-863-0025/1351 email: relliott@inovadx.com

6. Product Information and Classification

6.1 Proprietary and established names

Proprietary name(s):	NOVA Lite® DAPI ANCA (Ethanol) KitNOVA Lite® DAPI ANCA (Formalin) Kit
Common name:	antineutrophil cytoplasmic antibody kit
Classification name:	test system, antineutrophil cytoplasmic antibodies (ANCA)

6.2 Regulatory information

Regulation Description	Multiple autoantibodies immunological test system
Regulation Medical Specialty	Immunology
Review Panel	Immunology
Product Code	MOB

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Regulation Number	866.5660
Device Class	II

7. 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, 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 istoypes in human serum by manual fluorescence microscopy or with NOVA View Automated Fluorescence Microscope. The presence of ANCA, in conjunction with other serological, 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.

8. Indications for Use

Same as intended use.

9. Predicate device

NOVA Lite ANCA; 510k number: K961340

Item	NOVA Lite DAPI ANCA Ethanol and Formalin	Predicate Device
Intended use	Ethanol:NOVA Lite® DAPI ANCA (Ethanol) Kit isan indirect immunofluorescence assayfor the qualitative detection and semi-quantitative determination of anti-neutrophil cytoplasmic antibodies(ANCA) of IgG isotypes in humanserum by manual fluorescencemicroscopy or with NOVA ViewAutomated Fluorescence Microscope.The presence of ANCA, in conjunction	An indirect immunofluorescent testsystem utilizing ethanol-fixed humanneutrophil substrate slides fordetection of anti-neutrophilcytoplasmic antibodies (ANCA) inhuman serum. Detection of ANCAwhen used in conjunction with otherlaboratory and clinical findings aids inthe assessment of various systemicvasculitides, such as Wegener's
Item	NOVA Lite DAPI ANCA Ethanol and Formalinwith 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. Formalin: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 istoypes 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.	Predicate Device
Analyte	Anti- neutrophil cytoplasmic antibodies of IgG isotype	Anti- neutrophil cytoplasmic 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	Human neutrophil granulocytes, on 12-well slides, ethanol fixed or formalin fixed.	Human neutrophil granulocytes, on 12-well slides, ethanol fixed or formalin fixed.
Sample type	Serum	Serum
Sample dilution	1:20	1:20
Conjugate	FITC conjugated anti-human IgG (Fc specific) with added 4',6-diamidino-2-phenylindole (DAPI)	FITC conjugated anti-human IgG (Fc specific)
Item	NOVA Lite DAPI ANCA Ethanol and Formalin	Predicate Device
Additional dye in Conjugate	4',6-diamidino-2-phenylindole (DAPI)	None
Controls	C-ANCA, P-ANCA, and Negative Control	C-ANCA, P-ANCA, and Negative Control
Storage	2-8 °C	2-8 °C
Shelf life	18 months	18 months

10. Comparison matrix to predicate device

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11. Type of the product

Assay/reagents including controls. Qualitative and semi-quantitative. Device technology: indirect immunofluorescence.

12. Device description

Kit components:

. 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 PR3 and 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

13. Principle of the method and summary of the procedure

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 a DNAbinding 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 scanned with the NOVA View Automated Fluorescence Microscope. The resulting digital images are reviewed and interpreted from the computer monitor.

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ANCA positive samples exhibit an apple green fluorescence corresponding to areas of the cell where autoantibody has bound.

Manual interpretation

Ethanol ANCA:

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

C-ANCA: Coarse speckled cytoplasmic fluorescence, often with accentuated staining between the nuclear lobes. This pattern is characteristic for antibodies reacting with Proteinase 3 (PR3).

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

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 P-ANCA pattern.

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 and ANA usually become negative on formalin fixed substrate.

Formalin ANCA:

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 interpretation

When slides are analyzed by NOVA View, 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 measures the FITC light intensity of the cells that are included in the region. NOVA View reports the measured fluorescence intensity in units of Light Intensity Units (LIU).

NOVA View provides the trained operator with the acquired digital images and the following supportive information:

LIU value
Negative/positive classification
Pattern information

Patterns reported by NOVA View (Ethanol ANCA)
P	P-ANCA
C	C-ANCA
U	Unrecognized

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Patterns reported by NOVA View (Formalin ANCA)
N	Nuclear
C	Cytoplasmic
U	Unrecognized

NOVA View Single Well Titer

The Single Well Titer (SWT) is a software application that estimates the endpoint titer (i.e. the highest dilution that produces positive result) for wells with a positive reaction with Ethanol ANCA, based on the obtained LIU and pattern.

14. Analytical performance characteristics

14.1 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 software reported results obtained with the NOVA View Automated Fluorescence Microscope, such as Light Intensity Units (LIU), positive classification and pattern information.

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

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.

4+ Brilliant apple green fluorescence
3+ Bright apple green fluorescence
2+ Clearly distinguishable positive fluorescence
1+ Lowest specific fluorescence that enables the nuclear and/or cytoplasmic staining to be clearly differentiated from the background fluorescence

Pattern nomenclature: See Principle of method and summary of the procedure

14.2 Precision performance

14.2.1 Within laboratory imprecision

To assess the precision performance of the NOVA Lite DAPI ANCA Ethanol and ANCA Formalin Kit results, a study was performed by processing 3 negative and 13 positive samples: 7 anti-MPO and 6 anti-PR3 positive samples with various ANCA intensities, in three replicates, in 10 runs (2 runs per day),

{11}------------------------------------------------

resulting in 30 data points for each sample. The slides were read with NOVA View, and digital images were interpreted by the operator. Moreover, slides were read with manual microscope, too; i.e. three set of results were generated: NOVA View software interpretation, digital image reading results and manual reading results. Results were generated for within-run, between-day and total imprecision. 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.

The results of the above precision study are summarized in the Tables below.

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		Expected Result		Obtained Result
					NOVA View results			Manual results		Digital results
Sample	Specificity	n	Negative/Positive	Pattern	MeanLIU	%Negative	%Positive	Graderange(0-4+)	%Negative	%Positive	Graderange(0-4+)	%Negative	%Positive
1	Anti-MPO	30	Positive	P	1380.7	0.0%	100.0%	4	0.0%	100.0%	4	0.0%	100.0%
2	Anti-MPO	30	Positive	P	424.9	0.0%	100.0%	3	0.0%	100.0%	3-4	0.0%	100.0%
3	Anti-MPO	30	Positive	P	341.9	0.0%	100.0%	2-3	0.0%	100.0%	2-3	0.0%	100.0%
4	Anti-MPO	30	Positive	P	74.5	0.0%	100.0%	1-2	0.0%	100.0%	1	0.0%	100.0%
5	N/A	30	Negative	-	2.0	100.0%	0.0%	0	100.0%	0.0%	0	100.0%	0.0%
6	Anti-PR3	30	Positive	C	1056.6	0.0%	100.0%	4	0.0%	100.0%	4	0.0%	100.0%
7	Anti-PR3	30	Positive	C	388.2	0.0%	100.0%	2-3	0.0%	100.0%	2-4	0.0%	100.0%
8	Anti-PR3	30	Positive	C	80.0	0.0%	100.0%	2	0.0%	100.0%	1-2	0.0%	100.0%
9	Anti-PR3	30	Positive	C	34.4	13.0%	87.0%	1	0.0%	100.0%	1	0.0%	100.0%
10	Anti-PR3	30	Negative	-	3.4	100.0%	0.0%	0	100.0%	0.0%	0	100.0%	0.0%
11	N/A	30	Negative	-	2.3	100.0%	0.0%	0	100.0%	0.0%	0	100.0%	0.0%
12	Anti-MPO	30	Positive	P	658.8	0.0%	100.0%	4	0.0%	100.0%	4	0.0%	100.0%
13	Anti-MPO	30	Positive	P	563.3	0.0%	100.0%	3-4	0.0%	100.0%	3-4	0.0%	100.0%
14	Anti-MPO	30	Positive	P	850.8	0.0%	100.0%	4	0.0%	100.0%	4	0.0%	100.0%
15	Anti-PR3	30	Positive	C	866.1	0.0%	100.0%	4	0.0%	100.0%	4	0.0%	100.0%
16	N/A	30	Negative	-	2.0	100.0%	0.0%	0	100.0%	0.0%	0	100.0%	0.0%
New samples (close to cut-off)
17	Anti-MPO	30	Positive	P	36.4	13.3%	86.7%	1-2	0.0%	100.0%	0-1	6.7%	93.3%
18	Anti-MPO	30	Positive	P	13.1	86.7%	13.30%	1	0.0%	100.0%	0-1	40.0%	60.0%
19	Anti-PR3	30	Positive	C	29.9	30.0%	70.00%	1-2	0.0%	100.0%	1	0.0%	100.0%
20	Anti-MPO	30	Positive	P	18.7	63.3%	36.70%	1-2	0.0%	100.0%	0-1	23.3%	76.7%
21	Anti-PR3	30	Positive	C	70.0	0.00%	100.0%	1-2	0.0%	100.0%	1	0.0%	100.0%
22	Anti-PR3	30	Positive	C	57.0	13.3%	86.70%	1-2	0.0%	100.0%	1	0.0%	100.0%
23	Anti-PR3	30	Positive	C	57.1	23.3%	76.70%	1-2	0.0%	100.0%	0-2	10.0%	90.0%
24	Anti-PR3	30	Positive	C	26.0	33.3%	66.70%	1	0.0%	100.0%	0-1	13.3%	86.7%
25	Anti-MPO	30	Positive	P	15.1	76.7%	23.30%	1-2	0.0%	100.0%	0-1	30.0%	70.0%
26	Anti-MPO	30	Positive	P	16.0	70.00%	30.0%	1-2	0.0%	100.0%	0-1	40.0%	60.0%

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NOVA Lite® DAPI ANCA Ethanol and Formalin

Page 11 of 45

	Specificity	n	Expected Result	AverageLIU	Within run		Between run		Between day		Total (within lab)
Sample			Neg/Pos	Pattern		SD	%CV	SD	%CV	SD	%CV	SD	%CV
1	Anti-MPO	30	Positive	P	1380.7	178.9	13.0%	160.9	11.7%	0.0	0.0%	240.6	17.4%
2	Anti-MPO	30	Positive	P	424.9	56.8	13.4%	31.9	7.5%	0.0	0.0%	65.2	15.3%
3	Anti-MPO	30	Positive	P	341.9	11.2	3.3%	5.2	1.5%	1.7	0.5%	12.5	3.6%
4	Anti-MPO	30	Positive	P	74.5	18.2	24.5%	15.0	20.2%	29.9	40.1%	38.1	51.1%
5	N/A	30	Negative	-	2.0	0.2	9.0%	0.0	0.0%	0.0	0.0%	0.2	9.0%
6	Anti-PR3	30	Positive	C	1056.6	140.6	13.3%	126.4	12.0%	0.0	0.0%	189.1	17.9%
7	Anti-PR3	30	Positive	C	388.2	53.5	13.8%	41.6	10.7%	0.0	0.0%	67.8	17.5%
8	Anti-PR3	30	Positive	C	80.0	13.3	16.7%	19.8	24.8%	0.0	0.0%	23.9	29.9%
9	Anti-PR3	30	Positive	C	34.4	6.6	19.1%	12.2	35.4%	0.0	0.0%	13.8	40.2%
10	Anti-PR3	30	Negative	-	3.4	1.1	33.2%	1.7	48.3%	0.0	0.0%	2.0	58.7%
11	N/A	30	Negative	-	2.3	0.5	22.6%	0.0	0.0%	0.3	10.8%	0.6	25.0%
12	Anti-MPO	30	Positive	P	658.8	98.7	15.0%	0.0	0.0%	0.0	0.0%	98.7	15.0%
13	Anti-MPO	30	Positive	P	563.3	62.7	11.1%	85.6	15.2%	0.0	0.0%	106.1	18.8%
14	Anti-MPO	30	Positive	P	850.8	146.3	17.2%	0.0	0.0%	0.0	0.0%	146.3	17.2%
15	Anti-PR3	30	Positive	C	866.1	90.6	10.5%	0.0	0.0%	0.0	0.0%	90.6	10.5%
16	N/A	30	Negative	-	2.0	0.0	0.0%	0.0	0.0%	0.0	0.0%	0.0	0.0%
New samples (close to cut-off)
17	Anti-MPO	30	Positive	P	36.4	12.6	34.6%	10.9	29.9%	0.0	0.0%	16.7	45.7%
18	Anti-MPO	30	Positive	P	13.1	6.0	45.9%	1.4	10.7%	0.0	0.0%	6.2	47.1%
19	Anti-PR3	30	Positive	C	29.9	17.0	57.0%	10.8	36.3%	0.0	0.0%	20.2	67.6%
20	Anti-MPO	30	Positive	P	18.7	9.0	48.1%	5.7	30.4%	0.0	0.0%	10.7	56.9%
21	Anti-PR3	30	Positive	C	70.0	30.4	43.5%	14.3	20.3%	4.2	6.0%	33.9	48.4%
22	Anti-PR3	30	Positive	C	57.0	24.0	42.1%	17.5	30.7%	15.4	27.0%	33.4	58.7%
23	Anti-PR3	30	Positive	C	57.1	42.2	73.9%	19.5	34.2%	19.9	34.8%	50.6	88.6%
24	Anti-PR3	30	Positive	C	26.0	8.5	32.9%	8.5	32.9%	0.0	0.0%	12.1	46.5%
25	Anti-MPO	30	Positive	P	15.1	7.9	52.5%	0.0	0.0%	0.0	0.0%	7.9	52.5%
26	Anti-MPO	30	Positive	P	16.0	10.9	68.4%	0.0	0.0%	2.9	18.0%	11.3	70.7%

NOTE: NOVA View DAPI ANCA are qualitative assays and have to be confirmed by the user

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		Expected Result		Obtained Result
					NOVA View results				Manual results		Digital results
Sample	Specificity	n	Negative/Positive	Pattern	MeanLIU	%Negative	%Positive	Graderange(0-4+)	%Negative	%Positive	Graderange(0-4+)	%Negative	%Positive
1	Anti-MPO	30	Positive	C	788.8	0.0%	100.0%	4	0.0%	100.0%	4	0.0%	100.0%
2	Anti-MPO	30	Positive	C	218.7	0.0%	100.0%	2-3	0.0%	100.0%	2-3	0.0%	100.0%
3	Anti-MPO	30	Positive	C	127.4	0.0%	100.0%	2	0.0%	100.0%	1-2	0.0%	100.0%
4	Anti-MPO	30	Borderline	C/-	17.4	93.0%	7.0%	1	0.0%	100.0%	1	0.0%	100.0%
5	N/A	30	Negative	-	2.0	100.0%	0.0%	0	100.0%	0.0%	0	100.0%	0.0%
6	Anti-PR3	30	Positive	C	1104.9	0.0%	100.0%	4	0.0%	100.0%	4	0.0%	100.0%
7	Anti-PR3	30	Positive	C	491.2	0.0%	100.0%	3	0.0%	100.0%	3-4	0.0%	100.0%
8	Anti-PR3	30	Positive	C	131.4	0.0%	100.0%	2	0.0%	100.0%	1-2	0.0%	100.0%
9	Anti-PR3	30	Positive	C	128.5	0.0%	100.0%	1-2	0.0%	100.0%	1-2	0.0%	100.0%
10	Anti-PR3	30	Negative	-	2.8	100.0%	0.0%	0	100.0%	0.0%	0	100.0%	0.0%
11	N/A	30	Negative	-	2.0	100.0%	0.0%	0	100.0%	0.0%	0	100.0%	0.0%
12	Anti-MPO	30	Positive	C	406.7	0.0%	100.0%	3-4	0.0%	100.0%	3-4	0.0%	100.0%
13	Anti-MPO	30	Positive	C	263.8	0.0%	100.0%	2	0.0%	100.0%	2-3	0.0%	100.0%
14	Anti-MPO	30	Positive	C	517.7	0.0%	100.0%	3-4	0.0%	100.0%	3-4	0.0%	100.0%
15	Anti-PR3	30	Positive	C	1095.0	0.0%	100.0%	4	0.0%	100.0%	4	0.0%	100.0%
16	N/A	30	Negative	-	2.0	100.0%	0.0%	0	100.0%	0.0%	0	100.0%	0.0%
						New samples (close to cut-off)
17	Anti-MPO	30	Positive	C	28.8	80.0%	20.0%	1-2	0.0%	100.0%	0-1	16.7%	83.3%
18	Anti-MPO	30	Positive	C	32.5	43.3%	56.7%	1-2	0.0%	100.0%	0-1	3.3%	96.7%
19	Anti-PR3	30	Positive	C	16.8	90.0%	10.0%	1-2	0.0%	100.0%	0-1	56.7%	43.3%
20	Anti-MPO	30	Positive	C	36.7	30.0%	70.0%	1-2	0.0%	100.0%	1	0.0%	100.0%
21	Anti-MPO	30	Positive	C	22.9	76.7%	23.3%	1	0.00%	100.0%	0-1	13.3%	86.7%
22	Anti-MPO	30	Positive	C	24.3	86.7%	13.3%	1-2	0.0%	100.0%	0-1	3.3%	96.7%
23	Anti-PR3	30	Positive	C	26.9	70.0%	30.0%	1-2	0.0%	100.0%	0-1	13.3%	86.7%
24	Anti-PR3	30	Positive	C	20.3	86.7%	13.3%	1	0.0%	100.0%	0-1	16.7%	83.3%
25	Anti-PR3	30	Positive	C	34.0	50.0%	50.0%	1-2	0.0%	100.0%	0-1	10.0%	90.0%
26	Anti-PR3	30	Positive	C	35.7	56.7%	63.3%	1-2	0.00%	100.0%	0-1	3.3%	96.7%

ANCA Formalin repeatability and reproducibility

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			Expected Result			Within run		Between run		Between day		Total (within lab)
Sample	Specificity	n			Average
			Neg/Pos	Pattern	LIU	SD	%CV	SD	%CV	SD	%CV	SD	%CV
1	Anti-MPO	30	Positive	C	788.8	64.4	8.2%	66.3	8.4%	59.6	7.6%	110.0	13.9%
2	Anti-MPO	30	Positive	C	218.7	37.7	17.2%	14.5	6.6%	5.8	2.6%	40.8	18.7%
3	Anti-MPO	30	Positive	C	127.4	29.4	23.1%	11.4	9.0%	8.3	6.5%	32.6	25.6%
4	Anti-MPO	30	Borderline	C/-	17.4	9.7	56.1%	0.0	0.0%	4.1	23.8%	10.6	61.0%
5	N/A	30	Negative	-	2.0	0.0	0.0%	0.0	0.0%	0.0	0.0%	0.0	0.0%
6	Anti-PR3	30	Positive	C	1104.9	170.5	15.4%	0.0	0.0%	71.7	6.5%	185.0	16.7%
7	Anti-PR3	30	Positive	C	491.2	43.4	8.8%	8.1	1.7%	42.7	8.7%	61.5	12.5%
8	Anti-PR3	30	Positive	C	131.4	26.6	20.3%	0.0	0.0%	13.8	10.5%	30.0	22.8%
9	Anti-PR3	30	Positive	C	128.5	20.1	15.7%	29.7	23.1%	0.0	0.0%	35.9	27.9%
10	N/A	30	Negative	-	2.8	1.4	51.1%	0.0	0.0%	0.6	20.5%	1.6	55.1%
11	N/A	30	Negative	-	2.0	0.2	9.0%	0.1	2.6%	0.0	0.0%	0.2	9.3%
12	Anti-MPO	30	Positive	C	406.7	48.5	11.9%	10.4	2.6%	50.9	12.5%	71.1	17.5%
13	Anti-MPO	30	Positive	C	242.9	38.4	15.8%	7.0	2.9%	20.0	8.2%	43.8	18.0%
14	Anti-MPO	30	Positive	C	517.7	69.0	13.3%	28.9	5.6%	0.0	0.0%	74.8	14.4%
15	Anti-PR3	30	Positive	C	1095.0	187.2	17.1%	0.0	0.0%	44.4	4.1%	192.4	17.6%
16	N/A	30	Negative	-	2.0	0.0	0.0%	0.0	0.0%	0.0	0.0%	0.0	0.0%
New samples (close to cut-off)
17	Anti-MPO	30	Positive	C	28.8	7.8	27.2%	23.3	81.0%	0.0	0.0%	24.6	85.5%
18	Anti-MPO	30	Positive	C	32.5	7.5	23.2%	9.0	27.7%	0.0	0.0%	11.7	36.1%
19	Anti-PR3	30	Positive	C	16.8	8.3	49.7%	4.3	25.6%	0.0	0.0%	9.4	55.9%
20	Anti-MPO	30	Positive	C	36.7	6.8	18.4%	7.2	19.6%	0.0	0.0%	9.9	26.9%
21	Anti-MPO	30	Positive	C	22.9	5.5	23.8%	5.6	24.4%	0.6	2.4%	7.8	34.2%
22	Anti-MPO	30	Positive	C	24.3	11.3	46.5%	6.7	27.5%	0.0	0.0%	13.1	54.0%
23	Anti-PR3	30	Positive	C	26.9	13.0	48.5%	0.0	0.0%	0.0	0.0%	13.0	48.5%
24	Anti-PR3	30	Positive	C	20.3	7.7	38.1%	0.0	0.0%	5.8	28.6%	9.7	47.6%
25	Anti-PR3	30	Positive	C	34.0	17.1	50.4%	8.0	23.6%	0.0	0.0%	18.9	55.7%
26	Anti-PR3	30	Positive	C	35.7	13.2	37.0%	14.1	39.4%	0.0	0.0%	19.3	54.1%

NOTE: NOVA View DAPI ANCA are qualitative assays and have to be confirmed by the user

14.2.2 Between lots reproducibility

Lot to lot comparison study was performed on three reagent lots. Thirty-three clinically and/or analytically characterized samples were tested.

The following comparisons were made:

NOVA view output: qualitative agreement and pattern agreement. ●
Digital image reading: qualitative agreement, grade and pattern agreement .
. Manual image reading: qualitative agreement, grade and pattern agreement comparison.

Acceptance criteria:

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. Qualitative agreement: ≥ 90%
. Grade agreement: ≥ 90% within ± 1 reactivity grade
Pattern agreement: ≥ 90% for digital and manual reading ●

Qualitative Agreement, Ethanol ANCA:

Qualitative agreement in three-way comparisons ranged from 97.0% to 100.0%.

NOVA View Summary Table

NOVA View	Negative agreement (%)(95% CI)	Positive agreement (%)(95% CI)	Total agreement (%)(95% CI)
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)

Manual Summary Table

Manual	Negative agreement (%)(95% CI)	Positive agreement (%)(95% CI)	Total agreement (%)(95% CI)
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 (87.7 - 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)

Digital Summary Table

Digital	Negative agreement (%)(95% CI)	Positive agreement (%)(95% CI)	Total agreement (%)(95% CI)
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)

Qualitative Agreement, Formalin ANCA:

Total qualitative agreement in three-way comparisons ranged from 90.9% to 100.0%

NOVA View Summary Table

	Negative agreement (%)(95% CI)	Positive agreement (%)(95% CI)	Total agreement (%)(95% CI)
NOVA View
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)

Manual Summary Table

Manual	Negative agreement (%)(95% CI)	Positive agreement (%)(95% CI)	Total agreement (%)(95% CI)
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)

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

Digital Summary Table

Digital	Negative agreement (%)(95% CI)	Positive agreement (%)(95% CI)	Total agreement (%)(95% CI)
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)

Grade agreement, Ethanol ANCA:

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

	Manual grade comparison:							Digital grade comparison:
				Lot 018119							Lot 018119
Lot 17103	0	1	2	3	4	Total	Lot 017103	0	1	2	3	4	Total
0	6	0	0	0	0	6	0	6	0	0	0	0	6
1	0	3	1	0	0	4	1	0	3	0	0	0	3
2	0	0	8	0	0	8	2	0	2	5	0	0	7
3	0	0	1	6	1	8	3	0	0	3	3	0	6
4	0	0	0	0	7	7	4	0	0	0	1	10	11
Total	6	3	10	6	8	33	Total	6	5	8	4	10	33
within± one grade:		100%					within± one grade:		100%
				Lot 022657							Lot 022657
Lot 017103	0	1	2	3	4	Total	Lot 017103	0	1	2	3	4	Total
0	6	0	0	0	0	6	0	6	0	0	0	0	6
1	0	4	0	0	0	4	1	0	3	0	0	0	3
2	0	0	7	1	0	8	2	0	2	5	0	0	7
3	0	0	1	5	2	8	3	0	0	2	4	0	6
4	0	0	0	0	7	7	4	0	0	0	0	11	11
Total	6	4	8	6	9	33	Total	6	5	7	4	11	33
within± one grade:		100%					within± one grade:		100%
				Lot 022657							Lot 022657
Lot 18119	0	1	2	3	4	Total	Lot 018119	0	1	2	3	4	Total
0	6	0	0	0	0	6	0	6	0	0	0	0	6
1	0	3	0	0	0	3	1	0	5	0	0	0	5
2	0	1	8	1	0	10	2	0	0	6	2	0	8
3	0	0	0	5	1	6	3	0	0	1	2	1	4
4	0	0	0	0	8	8	4	0	0	0	0	10	10
Total	6	4	8	6	9	33	Total	6	5	7	4	11	33
within± one grade:		100%					within± one grade:		100%

Grade agreement, Formalin ANCA:

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

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

Manual grade comparison:

Lot 024112
Lot 018714	0	1	2	3	4	Total
0	7	0	0	0	0	7
1	0	4	0	0	0	4
2	0	1	8	2	0	11
3	0	0	0	4	2	6
4	0	0	0	0	5	5
Total	7	5	8	6	7	33
within± one grade:	100%
Lot RP0002
Lot 018714	0	1	2	3	4	Total
0	7	0	0	0	0	7
1	0	3	1	0	0	4
2	0	1	6	4	0	11
3	0	0	1	3	2	6
4	0	0	0	0	5	5
Total	7	4	8	7	7	33

Digital grade comparison:

	Lot 024112
Lot 018714	0	1	2	3	4	Total
0	9	0	0	0	0	9
1	0	4	2	0	0	6
2	0	1	3	5	0	9
3	0	0	0	1	1	2
4	0	0	0	2	5	7
Total	9	5	5	8	6	33
within± one grade:	100%
	Lot RP0002
Lot 018714	0	1	2	3	4	Total
0	7	2	0	0	0	9
1	1	3	2	0	0	6
2	0	0	5	4	0	9
3	0	0	1	1	0	2
4	0	0	0	0	7	7
Total	8	5	8	5	7	33

within± one grade:

100% within± one grade: 100% Lot RP0002 Lot RP0002 Lot 024112 Lot 024112 దు Total చ Total O O O O O ნ లు దు O దు రా ను O O Total Total within± one grade: 100% within± one grade: 100%

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

Pattern agreement, Ethanol ANCA:

Pattern agreement was 100% for both manual and digital reading. Pattern agreement for NOVA View software interpretation ranged from 71.9% to 75.8%. This agreement value includes positive/negative discrepancies, as well as discrepancy originating from NOVA View reporting Unrecognized pattern.

NOVA View pattern agreement						Manual pattern agreement					Digital pattern agreement
Lot 018119						Lot 018119					Lot 018119
Lot 017103	Neg	C	P	U	Total	Lot 017103	Neg	C	P	Total	Lot 017103	Neg	C	P	Total
Neg	6	0	0	0	6	Neg	6	0	0	6	Neg	6	0	0	6
C	1	2	0	6	9	C	0	13	0	13	C	0	13	0	13
P	0	0	12	0	12	P	0	0	14	14	P	0	0	14	14
U	0	0	1	4	5	Total	6	13	14	33	Total	6	13	14	33
Total	7	2	13	10	32	agreement: 100.0%					agreement: 100.0%
agreement: 75.0%
Lot 022657						Lot 022657					Lot 022657
Lot 017103	Neg	C	P	U	Total	Lot 017103	Neg	C	P	Total	Lot 017103	Neg	C	P	Total
Neg	6	0	0	0	6	Neg	6	0	0	6	Neg	6	0	0	6
C	1	5	0	3	9	C	0	13	0	13	C	0	13	0	13
P	0	0	12	1	13	P	0	0	14	14	P	0	0	14	14
U	0	2	1	2	5	Total	6	13	14	33	Total	6	13	14	33
Total	7	7	13	6	33	agreement: 100.0%					agreement: 100.0%
agreement: 75.8%
Lot 022657						Lot 022657					Lot 022657
Lot 018119	Neg	C	P	U	Total	Lot 018119	Neg	C	P	Total	Lot 018119	Neg	C	P	Total
Neg	7	0	0	0	7	Neg	6	0	0	6	Neg	6	0	0	6
C	0	1	0	1	2	C	0	13	0	13	C	0	13	0	13
P	0	1	11	1	13	P	0	0	14	14	P	0	0	14	14
U	0	5	1	4	10	Total	6	13	14	33	Total	6	13	14	33
Total	7	7	12	6	32	agreement: 100.0%					agreement: 100.0%
agreement: 71.9%

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

Pattern agreement, Formalin ANCA:

Pattern agreement ranged from 90.9% to 100% for both manual and digital reading. Pattern agreement for NOVA View software interpretation ranged from 81.8% to 87.9%. This agreement value includes positive/negative discrepancies, as well as discrepancy originating from NOVA View reporting Unrecognized pattern.

Lot 024112				Lot 024112				Lot 024112
Lot 018714	Neg	C	U	Total	Lot 018714	Neg	C	N	Total	Lot 018714	Neg	C	N	Total
Neg	11	0	0	11	Neg	7	0	0	7	Neg	9	0	0	9
C	0	16	1	17	C	0	25	0	25	C	0	24	0	24
U	1	2	2	5	N	0	0	1	1	N	0	0	0	0
Total	12	18	3	33	Total	7	25	1	33	Total	9	24	0	33
agreement:	87.9%				agreement:	100%				agreement:	100%
Lot RP0002					Lot RP0002					Lot RP0002
Lot 018714	Neg	C	U	Total	Lot 018714	Neg	C	N	Total	Lot 018714	Neg	C	N	Total
Neg	9	0	2	11	Neg	7	0	0	7	Neg	7	0	2	9
C	0	17	0	17	C	0	25	0	25	C	1	23	0	24
U	1	3	1	5	N	0	0	1	1	N	0	0	0	0
Total	10	20	3	33	Total	7	25	1	33	Total	8	23	2	33
agreement:	81.8%				agreement:	100%				agreement:	90.9%
Lot RP0002					Lot RP0002					Lot RP0002
Lot 024112	Neg	C	U	Total	Lot 024112	Neg	C	N	Total	Lot 024112	Neg	C	N	Total
Neg	10	0	2	12	Neg	7	0	0	7	Neg	7	0	2	9
C	0	18	0	18	C	0	25	0	25	C	1	23	0	24
U	0	2	1	3	N	0	0	1	1	N	0	0	0	0
Total	10	20	3	33	Total	7	25	1	33	Total	8	23	2	33
agreement:	87.9%				agreement:	100%				agreement:	90.9%

14.2.3 Between sites/instruments reproducibility

The same 287 clinically characterized samples were tested with both Ethanol ANCA and Formalin ANCA at three testing sites, using three different NOVA View instruments: in Inova's laboratory, and two external US clinical laboratories. The two external sites each also tested 100 routine clinical samples that ANCA testing was requested from. The composition of the sample cohorts and the results obtained by the three sites are shown below:

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

Site #1 (Inova)

Ethanol ANCA

Diagnosis	N	NOVA View pos		Manual pos		Digital pos
		#	%	#	%	#	%
ANCA Associated Vasculitis (AAV)	80	68	85%	71	89%	70	88%
Characterized MPO/PR3	49	47	96%	49	100%	49	100%
Infectious Disease	20	4	20%	11	55%	8	40%
Chronic Kidney Disease (CKD)	18	9	50%	13	72%	10	56%
Dermatitis	12	3	25%	4	33%	3	25%
Diabetes Type II	20	8	40%	8	40%	8	40%
Celiac (tTG IgA POS)	20	3	15%	4	20%	4	20%
Chronic Obstructive Pulmonary Disease (COPD)	20	8	40%	9	45%	9	45%
Rheumatoid Arthritis (RA)	15	12	80%	12	80%	12	80%
Crohn's Disease	11	6	55%	6	55%	6	55%
Ulcerative Colitis	15	4	27%	8	53%	5	33%
Allergy	7	2	29%	3	43%	2	29%
Total	287

Formalin ANCA

Diagnosis	N	NOVA View pos		Manual pos		Digital pos
		#	%	#	%	#	%
ANCA Associated Vasculitis (AAV)	80	66	83%	74	93%	71	89%
Characterized MPO/PR3	49	46	94%	49	100%	48	98%
Infectious Disease	20	0	0%	3	15%	0	0%
Chronic Kidney Disease (CKD)	18	0	0%	1	6%	1	6%
Dermatitis	12	0	0%	0	0%	0	0%
Diabetes Type II	20	0	0%	0	0%	1	5%
Celiac (tTG IgA POS)	20	0	0%	1	5%	1	5%
Chronic Obstructive Pulmonary Disease (COPD)	20	0	0%	2	10%	2	10%
Rheumatoid Arthritis (RA)	15	2	13%	3	20%	2	13%
Crohn's Disease	11	3	27%	2	18%	2	18%
Ulcerative Colitis	15	0	0%	2	13%	1	7%
Allergy	7	0	0%	1	14%	2	29%
Total	287

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

Site #2

Ethanol ANCA

Diagnosis	N	NOVA View pos		Manual pos		Digital pos
		#	%	#	%	#	%
ANCA Associated Vasculitis (AAV)	80	56	70%	60	75%	57	71%
Characterized MPO/PR3	49	46	94%	45	92%	46	94%
Infectious Disease	16*	2	13%	7	44%	3	19%
Chronic Kidney Disease (CKD)	18	8	44%	9	50%	8	44%
Dermatitis	12	3	25%	4	33%	3	25%
Diabetes Type II	20	3	15%	6	30%	4	20%
Celiac (tTG IgA POS)	20	2	10%	3	15%	2	10%
Chronic Obstructive Pulmonary Disease (COPD)	20	5	25%	7	35%	7	35%
Rheumatoid Arthritis (RA)	15	12	80%	13	87%	12	80%
Crohn's Disease	11	6	55%	6	55%	6	55%
Ulcerative Colitis	15	4	27%	7	47%	5	33%
Allergy	7	2	29%	2	29%	2	29%
Routine ANCA samples	100	46	46%	56	56%	49	49%
Total	383*

*4 samples were not tested due to sample depletion.

Formalin ANCA

Diagnosis	N	NOVA View pos		Manual pos		Digital pos
		#	%	#	%	#	%
ANCA Associated Vasculitis (AAV)	80	61	76%	64	80%	65	81%
Characterized MPO/PR3	49	45	92%	46	94%	47	96%
Infectious Disease	20	1	5%	5	25%	0	0%
Chronic Kidney Disease (CKD)	18	2	11%	1	6%	2	11%
Dermatitis	12	0	0%	0	0%	0	0%
Diabetes Type II	20	0	0%	1	5%	1	5%
Celiac (tTG IgA POS)	20	0	0%	1	5%	1	5%
Chronic Obstructive Pulmonary Disease (COPD)	20	1	5%	1	5%	1	5%
Rheumatoid Arthritis (RA)	15	1	7%	1	7%	1	7%
Crohn's Disease	11	3	27%	3	27%	2	18%
Ulcerative Colitis	15	0	0%	1	7%	0	0%
Allergy	7	1	14%	1	14%	1	14%
Routine ANCA samples	100	28	28%	31	31%	27	27%
Total	387

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

Site #3

Ethanol ANCA

Diagnosis	N	NOVA View pos		Manual pos		Digital pos
		#	%	#	%	#	%
ANCA Associated Vasculitis (AAV)	80	56	70%	58	73%	57	71%
Characterized MPO/PR3	49	46	94%	46	94%	47	96%
Infectious Disease	20	5	25%	4	20%	6	30%
Chronic Kidney Disease (CKD)	18	8	44%	9	50%	8	44%
Dermatitis	12	3	25%	4	33%	3	25%
Diabetes Type II	20	2	10%	4	20%	3	15%
Celiac (tTG IgA POS)	20	2	10%	3	15%	2	10%
Chronic Obstructive Pulmonary Disease (COPD)	20	6	30%	7	35%	7	35%
Rheumatoid Arthritis (RA)	15	11	73%	12	80%	12	80%
Crohn's Disease	11	6	55%	6	55%	6	55%
Ulcerative Colitis	15	5	33%	5	33%	5	33%
Allergy	7	2	29%	3	43%	2	29%
Routine ANCA samples	100	31	31%	37	37%	37	37%
Total	387

Formalin ANCA

Diagnosis	N	NOVA View pos		Manual pos		Digital pos
		#	%	#	%	#	%
ANCA Associated Vasculitis (AAV)	80	60	75%	62	78%	61	76%
Characterized MPO/PR3	49	42	86%	43	88%	44	90%
Infectious Disease	20	0	0%	2	10%	2	10%
Chronic Kidney Disease (CKD)	18	0	0%	0	0%	0	0%
Dermatitis	12	0	0%	0	0%	0	0%
Diabetes Type II	20	0	0%	1	5%	1	5%
Celiac (tTG IgA POS)	20	0	0%	1	5%	0	0%
Chronic Obstructive Pulmonary Disease (COPD)	20	2	10%	2	10%	2	10%
Rheumatoid Arthritis (RA)	15	1	7%	2	13%	1	7%
Crohn's Disease	11	2	18%	2	18%	2	18%
Ulcerative Colitis	15	0	0%	0	0%	0	0%
Allergy	7	0	0%	1	14%	1	14%
Routine ANCA samples	100	10	10%	10	10%	9	9%
Total	387

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

Results obtained on the 287 samples that were tested by each site were compared. Qualitative agreement, pattern agreement and grade agreement were calculated according to NOVA View software interpretation, manual reading and digital image reading.

Acceptance criteria:

Qualitative agreement: ≥ 85% .
. Grade agreement: ≥ 90% within ± 1 reactivity grade
. Pattern agreement: ≥ 80%, after excluding positive/negative discrepancies

Ethanol ANCA

Qualitative agreement:

NOVA View,n=287	Positive agreement (%)(95% CI)	Negative agreement (%)(95% CI)	Total agreement (%)(95% CI)
Site 1 vs Site 2	86.2 (80.3 - 90.6)	98.2 (93.8 - 99.5)	90.9 (87.1 - 93.7)
Site 1 vs Site 3	86.0 (80.1 - 90.4)	99.1 (95.1 - 99.8)	91.2 (87.3 - 93.9)
Site 2 vs Site 3	96.0 (91.5 - 98.2)	96.2 (91.5 - 98.4)	96.1 (93.2 - 97.8)

	Positive agreement (%)(95% CI)	Negative agreement (%)(95% CI)	Total agreement (%)(95% CI)
Manual, n=287	80.8 (74.8 - 85.7)	98.9 (93.9 - 99.8)	86.4 (82.0 - 89.9)
Site 1 vs Site 2	84.5 (78.8 - 88.9)	94.4 (87.5 - 97.6)	87.6 (83.3 - 91.0)
Site 1 vs Site 3	95.6 (91.2 - 97.9)	86.3 (79.1 - 91.3)	91.5 (87.7 - 94.2)
Site 2 vs Site 3

Digital, n=287	Positive agreement (%)(95% CI)	Negative agreement (%)(95% CI)	Total agreement (%)(95% CI)
Site 1 vs Site 2	85.5 (79.7 - 89.8)	100 (96.3 - 100)	90.6 (86.7 - 93.5)
Site 1 vs Site 3	84.7 (78.8 - 89.2)	100 (96.3 - 100)	90.1 (86.1 - 93.1)
Site 2 vs Site 3	96.2 (91.9 - 98.2)	96.1 (91.1 - 98.3)	96.1 (93.2 - 97.8)

Grade agreement:

Manual, 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%

{25}------------------------------------------------

Page	23 of 45
------	----------

Digital, 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:

NOVA View, n=287	Pattern agreement with pos/neg disagreement included	Pattern agreement without pos/neg disagreement excluded
Site 1 vs Site 2	84.0%	94.0%
Site 1 vs Site 3	81.0%	90.0%
Site 2 vs Site 3	90.0%	94.0%

	Pattern agreement with pos/neg disagreement included	Pattern agreement without pos/neg disagreement excluded
Manual, n=287	84.0%	98.0%
Site 1 vs Site 2	86.0%	98.0%
Site 1 vs Site 3	92.0%	100.0%
Site 2 vs Site 3

Digital, n=287	Pattern agreement with pos/negdisagreement included	Pattern agreement withoutpos/neg disagreement excluded
Site 1 vs Site 2	89.0%	98.0%
Site 1 vs Site 3	89.0%	98.0%
Site 2 vs Site 3	96.0%	100.0%

Formalin ANCA

Qualitative agreement:

NOVA View,n=287	Positive agreement (%)(95% CI)	Negative agreement (%)(95% CI)	Total agreement (%)(95% CI)
Site 1 vs Site 2	88.8 (81.8 - 93.3)	97.1 (93.3 - 98.7)	93.7 (90.3 - 96.0)
Site 1 vs Site 3	92.2 (85.9 - 95.9)	95.3 (91.0 - 97.6)	94.1 (90.7 - 96.3)
Site 2 vs Site 3	96.3 (90.9 - 98.6)	93.9 (89.3 - 96.5)	94.8 (91.6 - 96.8)

Manual, n=287	Positive agreement (%)	Negative agreement (%)	Total agreement (%)
	(95% CI)	(95% CI)	(95% CI)
Site 1 vs Site 2	81.9 (74.6 - 87.4)	98.0 (94.2 - 99.3)	90.2 (86.3 - 93.2)
Site 1 vs Site 3	86.2 (79.5 - 91.0)	96.0 (91.5 - 98.1)	91.3 (87.5 - 94.0)
Site 2 vs Site 3	92.2 (85.9 - 95.9)	89.5 (84.0 - 93.2)	90.6 (86.7 - 93.5)

{26}------------------------------------------------

Digital, n=287	Positive agreement (%)	Negative agreement (%)	Total agreement (%)
	(95% CI)	(95% CI)	(95% CI)
Site 1 vs Site 2	85.5 (78.5 - 90.5)	98.7 (95.4 - 99.6)	92.7 (89.1 - 95.2)
Site 1 vs Site 3	90.8 (84.7 - 94.7)	98.7 (95.4 - 99.6)	95.1 (92.0 - 97.1)
Site 2 vs Site 3	97.4 (92.5 - 99.1)	94.2 (89.7 - 96.8)	95.5 (92.4 - 97.3)

Grade agreement:

Manual, 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, 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, n=287	Pattern agreement with pos/neg disagreement included	Pattern agreement without pos/neg disagreement excluded
Site 1 vs Site 2	87.0%	93.0%
Site 1 vs Site 3	88.0%	93.0%
Site 2 vs Site 3	89.0%	94.0%

Manual, n=287	Pattern agreement with pos/neg disagreement included	Pattern agreement without pos/neg disagreement excluded
Site 1 vs Site 2	90.0%	100.0%
Site 1 vs Site 3	90.0%	98.0%
Site 2 vs Site 3	90.0%	99.0%

	Pattern agreement with pos/neg disagreement included	Pattern agreement without pos/neg disagreement excluded
Site 1 vs Site 2	92.0%	100.0%
Site 1 vs Site 3	94.0%	99.0%
Site 2 vs Site 3	94.0%	99.0%

{27}------------------------------------------------

14.2.4 Between operators reproducibility

Ten samples (2 negative, 4 P-ANCA and 4 C-ANCA positive) were tested at each site for 5 days in 5 replicates (25 data points per sample). Manual and digital reading was performed by two operators at each site, to assess between operators reproducibility.

Results are summarized below:

Ethanol ANCA, NOVA View Results

LIU:

		Expected result		NOVA View interpretation
Sample	n	neg/pos	pattern	Site #1Mean LIU	Site #2Mean LIU	Site #3Mean LIU
NVA 0224	25	pos	C	508.2	475.6	405.6
NVA 0232	25	pos	C	281.7	239.8	272.3
NVA 0226	25	pos	C	156.4	115.5	177.0
NVA 0225	25	pos	C	44.6	24.2	54.2
NVA 0206	25	pos	P	1257.2	1432.6	1403.6
NVA 0202	25	pos	P	589.3	664.3	673.7
NVA 0210	25	pos	P	226.0	274.8	280.3
NVA 0222	25	pos	P	163.5	125.5	155.7
NVA 0289	25	neg	n/a	3.2	2.0	2.6
NVA 0288	25	neg	n/a	3.1	2.1	2.7

Summary of SD and %CV:

Sample	n	Expected result		Average	Within Day		Between days		Within site		Between Site
		neg/pos	pattern	LIU	SD	%CV	SD	%CV	SD	%CV	SD	%CV
NVA 0224	25	pos	C	463.2	98.9	19.5%	76.4	15.1%	125.0	24.6%	0.0	0.0%
NVA 0232	25	pos	C	264.6	38.8	14.7%	34.7	13.1%	52.1	19.7%	13.5	5.1%
NVA 0226	25	pos	C	149.6	29.6	19.8%	33.3	22.3%	44.6	29.8%	26.9	18.0%
NVA 0225	25	pos	C	41.0	9.2	22.5%	12.3	30.0%	15.4	37.5%	14.2	34.6%
NVA 0206	25	pos	P	1364.4	133.4	9.8%	135.7	9.9%	190.3	13.9%	66.7	4.9%
NVA 0202	25	pos	P	642.4	88.8	13.8%	80.7	12.6%	120.0	18.7%	22.9	3.6%
NVA 0210	25	pos	P	260.4	36.1	13.9%	21.2	8.1%	41.9	16.1%	27.4	10.5%
NVA 0222	25	pos	P	148.2	31.7	21.4%	30.4	20.5%	43.9	29.6%	13.4	9.0%
NVA 0289	25	neg	n/a	2.6	0.9	36.9%	0.6	22.8%	1.1	43.4%	0.5	18.7%
NVA 0288	25	neg	n/a	2.6	1.2	44.3%	0.7	25.5%	1.3	51.1%	0.3	11.4%

{28}------------------------------------------------

Ethanol ANCA, manual reading Qualitative agreement:

		Expected result		Manual reading
				Site #1		Site #2		Site #3
				Reader #1	Reader #2	Reader #1	Reader #2	Reader #1	Reader #2
Sample	n	neg/pos	pattern	% neg	% pos	% neg	% pos	% neg	% pos	% neg	% pos	% neg	% pos	% neg	% pos
1	25	pos	C	0%	100%	0%	100%	0%	100%	0%	100%	0%	100%	0%	100%
2	25	pos	C	0%	100%	0%	100%	0%	100%	0%	100%	0%	100%	0%	100%
3	25	pos	C	0%	100%	0%	100%	0%	100%	0%	100%	0%	100%	0%	100%
4	25	pos	C	0%	100%	0%	100%	56%	44%	0%	100%	0%	100%	0%	100%
5	25	pos	P	0%	100%	0%	100%	0%	100%	0%	100%	0%	100%	0%	100%
6	25	pos	P	0%	100%	0%	100%	0%	100%	0%	100%	0%	100%	0%	100%
7	25	pos	P	0%	100%	0%	100%	0%	100%	0%	100%	0%	100%	0%	100%
8	25	pos	P	0%	100%	0%	100%	0%	100%	0%	100%	0%	100%	0%	100%
9	25	neg	n/a	100%	0%	100%	0%	100%	0%	100%	0%	100%	0%	100%	0%
10	25	neg	n/a	100%	0%	100%	0%	100%	0%	100%	0%	100%	0%	100%	0%

Grade agreement:

Sample	n	neg/pos	pattern	Site #1		Site #2		Site #3
				Reader #1	Reader #2	Reader #1	Reader #2	Reader #1	Reader #2
1	25	pos	C	3.4	3.0	2.8	3.4	3.9	3.0
2	25	pos	C	2.9	2.0	1.7	2.3	2.0	1.7
3	25	pos	C	1.9	1.4	1.2	1.6	1.5	1.1
4	25	pos	C	1.0	1.0	0.4	1.0	1.0	1.0
5	25	pos	P	4.0	4.0	4.0	4.0	4.0	3.8
6	25	pos	P	3.0	3.1	3.2	3.2	3.1	2.2
7	25	pos	P	1.9	1.8	2.0	2.0	2.0	1.6
8	25	pos	P	1.6	2.0	1.0	1.7	2.0	1.2
9	25	neg	n/a	0.0	0.0	0.0	0.0	0.0	0.0
10	25	neg	n/a	0.0	0.0	0.0	0.0	0.0	0.0

{29}------------------------------------------------

Ethanol ANCA, digital reading Qualitative agreement:

		Expected result		Digital reading
Sample	n	neg/pos	pattern	Site #1		Site #2		Site #3
				Reader #1	Reader #2	Reader #1	Reader #2	Reader #1	Reader #2
				% neg	% pos	% neg	% pos	% neg	% pos	% neg	% pos
1	25	pos	C	0%	100%	0%	100%	0%	100%	0%	100%
2	25	pos	C	0%	100%	0%	100%	0%	100%	0%	100%
3	25	pos	C	0%	100%	0%	100%	0%	100%	0%	100%
4	25	pos	C	0%	100%	0%	100%	8%	92%	4%	96%
5	25	pos	P	0%	100%	0%	100%	0%	100%	0%	100%
6	25	pos	P	0%	100%	0%	100%	0%	100%	0%	100%
7	25	pos	P	0%	100%	0%	100%	0%	100%	0%	100%
8	25	pos	P	0%	100%	0%	100%	0%	100%	0%	100%
9	25	neg	n/a	100%	0%	100%	0%	100%	0%	100%	0%
10	25	neg	n/a	100%	0%	100%	0%	100%	0%	100%	0%

Grade agreement:

Sample	n	neg/pos	pattern	Site #1		Site #2		Site #3
				Reader #1	Reader #2	Reader #1	Reader #2	Reader #1	Reader #2
1	25	pos	C	3.6	3.5	3.2	4.0	4.0	3.3
2	25	pos	C	3.0	2.6	2.0	2.2	2.6	1.5
3	25	pos	C	2.0	2.0	1.4	1.8	2.1	1.2
4	25	pos	C	1.0	1.0	0.9	1.0	1.0	0.7
5	25	pos	P	4.0	4.0	4.0	4.0	4.0	4.0
6	25	pos	P	3.6	4.0	3.6	3.4	4.0	3.5
7	25	pos	P	1.9	2.0	2.0	2.1	2.0	1.7
8	25	pos	P	1.7	1.8	1.1	1.2	1.3	1.0
9	25	neg	n/a	0.0	0.0	0.0	0.0	0.0	0.0
10	25	neg	n/a	0.0	0.0	0.0	0.0	0.0	0.0

Ethanol ANCA

Agreement between Operator 1 and Operator 2 at each site:

% Overall Agreement (Positive/Negative) between operators (per site) for Ethanol
	Inova	Site #2	Site #3
Manual Reading	100.0%	100.0%	94.4%
Digital Image Reading	100.0%	97.2%	98.8%

{30}------------------------------------------------

Formalin ANCA, NOVA View Results

LIU:

			NOVA View interpretation
		Expected result		Site #1	Site #2	Site #3
				Mean LIU	Mean LIU	Mean LIU
Sample	n	neg/pos	pattern
NVA 0224	25	pos	C	598.4	657.4	721.9
NVA 0232	25	pos	C	238.8	236.3	256.0
NVA 0226	25	pos	C	174.0	178.6	194.6
NVA 0225	25	pos	C	123.7	113.0	141.0
NVA 0206	25	pos	C	376.5	327.2	387.9
NVA 0202	25	pos	C	310.7	263.4	319.3
NVA 0210	25	pos	C	176.6	186.3	189.6
NVA 0222	25	borderline	C/-	11.0	8.0	8.2
NVA 0289	25	neg	n/a	2.0	2.0	2.0
NVA 0288	25	neg	n/a	2.1	2.0	2.0

Summary of SD and %CV:

Sample	n	Expected result		Average	Within Day		Betweendays		Within site		Between Site
		neg/pos	pattern	LIU	SD	%CV	SD	%CV	SD	%CV	SD	%CV
NVA 0224	25	pos	C	659.2	88.6	13.4%	0.0	0.0%	88.6	13.4%	59.2	9.0%
NVA 0232	25	pos	C	243.7	35.1	14.4%	6.7	2.7%	35.8	14.7%	7.6	3.1%
NVA 0226	25	pos	C	182.4	22.9	12.6%	18.1	9.9%	29.2	16.0%	5.5	3.0%
NVA 0225	25	pos	C	125.9	12.8	10.1%	11.2	8.9%	17.0	13.5%	13.0	10.3%
NVA 0206	25	pos	C	363.9	43.9	12.1%	35.6	9.8%	56.5	15.5%	26.6	7.3%
NVA 0202	25	pos	C	297.8	36.1	12.1%	41.0	13.8%	54.6	18.3%	22.7	7.6%
NVA 0210	25	pos	C	184.6	28.6	15.5%	4.9	2.6%	29.0	15.7%	0.0	0.0%
NVA 0222	25	borderline	C/-	9.3	6.4	69.3%	2.1	22.8%	6.8	73.0%	1.3	13.7%
NVA 0289	25	neg	n/a	2.0	0.2	8.1%	0.0	0.0%	0.2	8.1%	0.0	0.0%
NVA 0288	25	neg	n/a	2.0	0.2	9.8%	0.0	0.0%	0.2	9.8%	0.1	3.1%

{31}------------------------------------------------

Formalin ANCA, manual reading
Qualitative agreement:

		Expected result		Manual reading
				Site #1		Site #2		Site #3
Sample	n	neg/pos	pattern	Reader #1% neg	Reader #2% pos	Reader #1% neg	Reader #2% pos	Reader #1% neg	Reader #2% pos
1	25	pos	C	0%	100%	0%	100%	0%	100%
2	25	pos	C	0%	100%	0%	100%	0%	100%
3	25	pos	C	0%	100%	0%	100%	0%	100%
4	25	pos	C	0%	100%	0%	100%	0%	100%
5	25	pos	C	0%	100%	0%	100%	0%	100%
6	25	pos	C	0%	100%	0%	100%	0%	100%
7	25	pos	C	0%	100%	0%	100%	0%	100%
8	25	borderline	C/-	0%	100%	8%	92%	60%	40%
9	25	neg	n/a	100%	0%	100%	0%	100%	0%
10	25	neg	n/a	100%	0%	100%	0%	100%	0%

Grade agreement:

	Expected result		Manual reading grades
	Sample	n	neg/pos	pattern	Site #1Reader #1Average grade		Site #2Reader #1Average grade		Site #3Reader #1Average grade
1	25	pos	C	4.0	4.0	4.0	4.0	4.0	3.9
2	25	pos	C	3.0	3.2	2.4	3.2	3.0	2.6
3	25	pos	C	2.1	2.5	2.5	2.5	3.0	2.0
4	25	pos	C	2.0	2.2	1.3	1.9	2.4	1.6
5	25	pos	C	3.6	3.5	3.2	3.3	4.0	2.9
6	25	pos	C	3.0	3.0	2.4	3.2	4.0	2.6
7	25	pos	C	2.0	2.0	2.0	2.4	3.0	1.6
8	25	borderline	C/-	1.0	0.9	0.4	1.0	1.0	1.0
9	25	neg	n/a	0.0	0.0	0.0	0.0	0.0	0.0
10	25	neg	n/a	0.0	0.0	0.0	0.0	0.0	0.0

{32}------------------------------------------------

Formalin ANCA, digital reading Qualitative agreement:

				Digital reading
		Expected result		Site #1		Site #2		Site #3
				Reader #1	Reader #2	Reader #1	Reader #2	Reader #1	Reader #2
Sample	n	neg/pos	pattern	%neg	% pos	%neg	% pos	%neg	% pos	%neg	% pos
1	25	pos	C	0%	100%	0%	100%	0%	100%	0%	100%
2	25	pos	C	0%	100%	0%	100%	0%	100%	0%	100%
3	25	pos	C	0%	100%	0%	100%	0%	100%	0%	100%
4	25	pos	C	0%	100%	0%	100%	0%	100%	4%	96%
5	25	pos	C	0%	100%	0%	100%	0%	100%	0%	100%
6	25	pos	C	0%	100%	0%	100%	0%	100%	0%	100%
7	25	pos	C	0%	100%	0%	100%	0%	100%	0%	100%
8	25	borderline	C/-	28%	72%	48%	52%	92%	8%	100%	0%
9	25	neg	n/a	100%	0%	100%	0%	100%	0%	100%	0%
10	25	neg	n/a	100%	0%	100%	0%	100%	0%	100%	0%

Grade agreement:

		Expected result		Digital reading grades
				Site #1		Site #2		Site #3
Sample	n	neg/pos	pattern	Reader #1	Reader #2	Reader #1	Reader #2	Reader #1	Reader #2
				Average grade		Average grade		Average grade
1	25	pos	C	4.0	4.0	4.0	4.0	4.0	3.2
2	25	pos	C	3.0	3.0	2.2	3.0	3.4	2.0
3	25	pos	C	2.2	2.0	2.0	2.1	2.6	1.0
4	25	pos	C	1.4	1.4	1.4	1.6	2.4	1.0
5	25	pos	C	3.8	3.1	3.0	3.0	4.0	3.0
6	25	pos	C	3.1	2.8	2.2	2.5	3.8	2.1
7	25	pos	C	2.0	2.0	1.8	2.0	2.6	1.0
8	25	borderline	C/-	0.7	0.5	0.1	0.9	0.0	0.0
9	25	neg	n/a	0.0	0.0	0.0	0.0	0.0	0.0
10	25	neg	n/a	0.0	0.0	0.0	0.0	0.0	0.0

{33}------------------------------------------------

Formalin ANCA

Agreement between Operator 1 and Operator 2 at each site:

% Overall Agreement (Positive/Negative) between operators (per site) for Formalin
	Inova	Site #2	Site #3
Manual Reading	99.2%	100.0%	94.0%
Digital Image Reading	97.2%	99.6%	91.6%

14.3 Linearity and Analytical Measuring Range (AMR)

N/A

14.4 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, RF IgM, Human Immunoglobulin (IgG), Rituximab, Methylprednisolone, Cyclophosphamide, Methotrexate, Azathioprine was assessed using the following materials and concentrations. The Table below contains all three concentration levels that were tested:

Interfering substance	ManufacturerPart Number	Lot number	Concentration #1tested	Concentration #2tested	Concentration #3tested
Bilirubin, conjugated	EMD 201102	DU3038000	100 mg/dL	50 mg/dL	25 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/dL	56 mg/dL
RF IgM	QC #16	ZG0024783	56 IU/mL	39.2 IU/mL	28.02 IU/mL

Interfering substance	ManufacturerPart Number	Lot number	Concentration#1 tested	Concentration #2tested	Concentration #3
Human Immunoglobulin (IgG)	16-16-090701	1G2012-05	70 mg/mL	35 mg/mL	17.5 mg/mL
Rituximab	SAP-10063468	n/a	7.6 mg/mL	3.8 mg/mL	1.9 mg/mL
Methylprednisolone	1435003	I0E170	0.85 mg/mL	0.425 mg/mL	0.213 mg/mL
Cyclophosphamide	PHR1404	LRAA2198	4.1 mg/mL	2.05 mg/mL	1.025 mg/mL
Methotrexate	1414003	R020L0	0.01 mg/mL	0.005 mg/mL	0.0025 mg/mL
Azathioprine	PHR1282	LRAA9079	0.03 mg/mL	0.015 mg/mL	0.0075 mg/mL

Five specimens were tested (one negative, one low anti-MPO positive, one strong anti-MPO positive, one low anti-PR3 positive, one strong anti-PR3 positive). Interfering substances (see tables above) were spiked into every specimen at three different concentrations (see tables above) in 10% of total specimen

{34}------------------------------------------------

volume, and the resulting samples were assessed in triplicates according to the standard protocol (diluted in 1:20 and processed on Ethanol ANCA and Formalin ANCA 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. All 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.

Acceptance criteria:

Grades obtained on samples with interfering substances are within ± 1 reactivity . grade of those obtained on the control samples, spiked with diluent.
No interference was detected with bilirubin up to 100 mg/dL, hemoglobin up to 200 mg/dL, triglycerides up to 1000 mg/dL, cholesterol up to 224.3 mg/dL, RF IgM up to 56 IU/mL, Human Immunoglobulin up to 35 mg/dL, Rituximab up to 7.6 mg/mL, Methy|prednisolone up to 0.85 mg/mL, Cyclophosphamide up to 4.1 mg/mL, Methotrexate up to 0.01 mg/mL and Azathioprine up to 0.03 mg/mL.

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

14.5 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 software interpretation and digital image interpretation, and 8%, 8% and 11% cross-reactivity was shown on Formalin-fixed slides, respectively:

Diagnosis	N	NOVA View pos		Manual pos		Digital pos
Infectious Disease	40	14	35%	14	35%	14	35%
Autoimmune thyroiddisease	15	14	93%	13	87%	13	87%
Celiac (tTG IgA POS)	61	8	13%	9	15%	8	13%
Rheumatoid Arthritis (RA)	35	2	6%	3	9%	2	6%
Total	151	38	25%	39	26%	37	25%

Ethanol ANCA cross-reactivity with autoimmune and infectious agent antibodies:

Formalin ANCA cross-reactivity with autoimmune and infectious agent antibodies:

Diagnosis	N	NOVA View pos		Manual pos	Digital pos
Infectious Disease	40	3	8%	2 5%	3	8%
Autoimmune thyroid disease	15	6	40%	6 40%	7	47%
Celiac (tTG IgA POS)	61	3	5%	3 5%	4	7%

{35}------------------------------------------------

NOVA Lite® DAPI ANCA Ethanol and Formalin

Rheumatoid Arthritis (RA)	35	0	0%	1	3%	2	6%
Total	151	12	8%	12	8%	16	11%

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 or in the samples.

Therefore 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 software interpretation, manual interpretation and digital image interpretation, and 24%, 32% and 24% cross-reactivity was shown on Formalin-fixed slides, respectively:

ANA positive sample	Number and % of Ethanol ANCA positive samples
	NOVA View	Manual	Digital
Homogeneous (n=5)	5100%	5100%	5100%
Speckled (n=5)	480%	480%	480%
Centromere (n=5)	5100%	5100%	5100%
Nucleolar (n=5)	360%	360%	360%
Nuclear dots (n=5)	240%	360%	360%
Total	1976%	2080%	2080%

Ethanol ANCA cross-reactivity with ANA:

Formalin ANCA cross-reactivity with ANA:

ANA positive sample	Number and % of Formalin ANCA positive samples
	NOVA View	Manual	Digital
Homogeneous (n=5)	4 80%	4 80%	4 80%
Speckled (n=5)	1 20%	1 20%	1 20%
Centromere (n=5)	1 20%	1 20%	1 20%
Nucleolar (n=5)	0 0%	1 20%	0 0%
Nuclear dots (n=5)	0 0%	1 20%	0 0%
Total	6 24%	8 32%	6 24%

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

Ethanol ANCA:

ANA positive samples may react with the nuclei of ethanol-fixed neutrophils, masking or mimicking ANCA. Positive IIF results should be confirmed by antigen specific solid phase assay for anti-MPO and anti-PR3.

Formalin ANCA:

{36}------------------------------------------------

ANA positive samples may show weak nuclear fluorescence on formalin fixed neutrophil granulocytes. 15. Cutoff

The serum dilution of 1:20 was selected to provide optimal clinical sensitivity and specificity, and it is the same as that of the predicate device.

Sera from 90 apparently healthy subjects (44 females, 46 males) and from 20 patients with infectious diseases (10 females, 5 unknown) were tested. The number of positive results is shown in the Table below:

	NOVA View softwareinterpretation	Manual reading	Digital reading
Ethanol ANCA	13	15	14
Formalin ANCA	4	2	2

The performance of this serum dilution has been validated as described in sections "Clinical performance" and "Expected values".

16. Comparison with the predicate device

16.1 Conjugate comparison

The conjugate (P/N 508102) is the same that is used in the NOVA Lite DAPI ANA Kit (k 155055). It is the same conjugate as that of the predicate device (P/N 508113), but with blue fluorescent dye DAPI (4',6diamidino-2-phenylindole) added.

To demonstrate the equivalent performance of the conjugate with and without DAPI, a comparison study was performed.

Comparison was performed on 36 specimens (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 ANCA Ethanol and ANCA Formalin reagents. Results were manually interpreted. Acceptance criteria:

. Qualitative agreement: ≥ 90%
Grade agreement: ≥ 90% within ± 1 reactivity grade

Moreover, endpoint titration was performed on 6 positive samples with both FITC IgG Conjugate (508113) and FITC IgG Conjugate with DAPI (508102).

Acceptance criteria:

. The endpoint dilution is within ±1 dilution step between the two conjugates
The qualitative agreement and the grade agreement between the result obtained with the predicate and the new conjugate was 100% on both Ethanol and Formalin ANCA slides.

{37}------------------------------------------------

	Grade 508102
Ethanol	0	2	3	4	Total
Grade 508113
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	Grade 508102
Grade 508113	0	1	2	3	4	Total
0	20	0	0	0	0	20
1	0	2	0	0	0	2
2	0	0	1	1	0	2
3	0	0	0	1	0	1
4	0	0	0	1	10	11
Total	20	2	1	3	10	36

Endpoint titers obtained with the new conjugate were within ±1 dilution step of those obtained with the predicate conjugate:

	Endpoint titer, Ethanol		Endpoint titer, Formalin
Sample ID	508113	508102	508113	508102
pANCA pos (PS0031/120401)	320	320	320	320
pANCA pos (PS0031/720005)	640	640	320	320
pANCA pos (PS0031/920124)	1280	1280	320	320
cANCA pos (PS0030/020187)	5120	5120	2560	2560
cANCA pos (PS0030/070828)	1280	2560	1280	1280
cANCA pos (PS0030/090263)	1280	1280	2560	2560

16.2 Method comparison

Results that were obtained with the NOVA Lite DAPI ANCA (Ethanol) Kit and NOVA Lite DAPI ANCA (Formalin) Kit by NOVA View software interpretation, digital image reading and manual reading were compared to those obtained with the manual interpretation of the predicate device. Study includes 100 samples (50 P-ANCA and 50 C-ANCA) and disease control groups (infectious disease, Systemic Lupus Erythemtosus, Progressive Systemic Sclerosis, Rheumatoid arthritis and Chronic Kidney Disease) Qualitative agreement, pattern agreement and grade agreement were calculated between NOVA View software interpretation, manual reading and digital image reading.

{38}------------------------------------------------

Acceptance criteria:

Qualitative agreement: ≥ 80% (this acceptance criteria only applies to manual and digital because NOVA View always has to be confirmed by digital interpretation)
Grade agreement: ≥ 90% within ± 1 reactivity grade ●
Pattern agreement: ≥ 80% between manual and digital interpretation.

The composition of the internal validation study is shown below:

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

Agreement results are summarized below for Inova Only:

Ethanol ANCA

Qualitative agreement:

n= 267	Positiveagreement (%)(95% CI)	Negativeagreement (%)(95% CI)	Total agreement(%) (95% CI)
708298 manual vs704338 manual	91.8 (87.6-94.7)	91.2 (77.0-97.0)	91.8 (87.8-94.5)
708298 manual vs 704338 digital	80.3 (74.7-84.9)	97.1 (85.1-99.5)	82.4 (77.4-86.5)
708298 manual vs 704338 NOVA View	76.4 (70.5-81.4)	97.1 (85.1-99.5)	79.0 (73.7-83.5)

Grade agreement:

	708298 Manual Grade
704338 ManualGrade	0	1+	2+	3+	4+	Total
0	31	10	9	0	0	50
1+	3	50	10	1	0	64
2+	0	5	46	13	1	65
3+	0	0	4	21	7	32
4+	0	1	1	8	46	56
Total	34	66	70	43	54	267
Within ± 2 grades	99.6%

{39}------------------------------------------------

	708298 Manual Grade
704338 DigitalGrade	0	1+	2+	3+	4+	Total
0	33	35	11	0	0	79
1+	1	28	25	3	0	57
2+	0	2	24	11	1	38
3+	0	0	8	18	4	30
4+	0	।	2	ਹ ਹ	49	રૂડે જિલ્લામાં આવેલું એક ગામનાં મુખ્યત્વે ખેત-ઉપર તાલુકામાં આવેલું એક ગામનાં મુખ્યત્વે ખેત-ઉપર તાલુકામાં આવેલું એક ગામનાં મુખ્યત્વે ખાતે ખાતમજૂરી તેમ જ પશુપાલન છે. આ ગામમા
Total	34	୧୧	70	43	54	267

Within ± 2 grades

99.6%

Pattern agreement:

n=267	Pattern agreement withpos/neg disagreement
708298 Manual vs 704338 Manual	89.9%
708298 Manual vs 704338 Digital	80.1%

Formalin ANCA

Qualitative agreement:

n= 267	Positive agreement(%) (95% CI)	Negative agreement(%) (95% CI)	Total agreement (%)(95% CI)
708297 manualvs704337 manual	94.4 (89.8-97.0)	90.5 (83.4-94.7)	92.9 (89.2-95.4)
708297 manual vs704337 digital	79.0 (72.1-84.6)	99.0 (94.8-99.8)	86.9 (82.3-90.4)
708297 manual vs704337 NOVA View	72.2 (64.9-78.5)	97.1 (91.9-99.0)	82.0 (77.0-86.2)

Grade agreement:

	708298 Manual Grade
704338 ManualGrade	0	1+	2+	3+	4+	Total
0	95	9	0	0	0	104
1+	9	36	12	1	0	58
2+	1	4	36	8	2	51
3+	0	0	1	23	4	28
4+	0	0	0	1	25	26
Total	105	49	49	33	31	267
Within ± 2 grades	100%

{40}------------------------------------------------

	708298 Manual Grade
704338 Digital Grade	0	1+	2+	3+	4+	Total
0	104	27	7	0	0	138
1+	1	21	13	1	0	36
2+	0	1	23	6	1	31
3+	0	0	6	21	3	30
4+	0	0	0	5	27	32
Total	105	49	49	33	31	267
Within ± 2 grades	100%

Within ± 2 grades

Pattern agreement:

n=267	Pattern agreement with pos/neg disagreement
708297 Manual vs 704337 Manual	92.1%
708297 Manual vs 704337 Digital	86.9%

17. Clinical performance

Prevalence of ANCA positivity on Formalin and Ethanol slides for NOVA View manual reading and digital interpretation.

Diagnosis	N	NOVA View Pos (% pos)		Manual pos (% pos)		Digital pos (% pos)
		Ethanol	Formalin	Ethanol	Formalin	Ethanol	Formalin
ANCA Associated Vasculitis (AAV)**	185	111(60%)	113 (61%)	131 (71%)	139 (75%)	129 (70%)	134 (72%)
Characterized MPO/PR3	59	57 (97%)	56 (95%)	59 (100%)	59 (100%)	59 (100%)	58 (98%)
Infectious Disease	40*	5 (13%)	4 (10%)	12 (30%)	6 (15%)	9 (23%)	5 (13%)
Autoimmune thyroid disease	15*	14 (93%)	6 (40%)	13 (87%)	6 (40%)	13 (87%)	7 (47%)
Chronic Kidney Disease (CKD)	18*	9 (50%)	0 (0%)	13 (72%)	1 (6%)	10 (56%)	1 (6%)
Dermatitis	12*	3 (25%)	0 (0%)	4 (33%)	0 (0%)	3 (25%)	0 (0%)
Diabetes Type II	20*	8 (40%)	0 (0%)	8 (40%)	0 (0%)	8 (40%)	1 (5%)
Celiac (tTG IgA POS)	61*	11 (18%)	5 (8%)	14 (23%)	4 (7%)	13 (21%)	3 (5%)
Chronic Obstructive Pulmonary Disease (COPD)	20*	8 (40%)	0 (0%)	9 (45%)	2 (10%)	9 (45%)	2 (10%)
Rheumatoid Arthritis (RA)	35*	25 (71%)	6 (17%)	26 (74%)	5 (14%)	25 (71%)	5 (14%)
Crohn's Disease	11*	6 (55%)	3 (27%)	6 (55%)	2 (18%)	6 (55%)	2 (18%)
Ulcerative Colitis	15*	4 (27%)	0 (0%)	8 (53%)	2 (13%)	5 (33%)	1 (7%)
Allergy	7*	2 (29%)	0 (0%)	3 (43%)	1 (14%)	2 (29%)	2 (29%)
Apparently healthy	89	4 (4%)	0 (0%)	8 (9%)	0 (0%)	4 (4%)	0 (0%)
Progressive systemic sclerosis	25*	14 (56%)	1 (4%)	18 (72%)	3 (12%)	14 (56%)	2 (8%)
Sinusitis	18*	1 (6%)	0 (0%)	5 (28%)	1 (6%)	2 (11%)	0 (0%)
Systemic Lupus Erythematosus	23*	16 (70%)	4 (17%)	18 (78%)	9 (39%)	15 (65%)	5 (22%)
Total	653

*used for specificity calculation

{41}------------------------------------------------

		NOVA View Pos (% pos)		Manual pos (% pos)		Digital pos (% pos)
Diagnosis	N	Ethanol	Formalin	Ethanol	Formalin	Ethanol	Formalin
GPA	113	53 (47%)	67 (59%)	67 (59%)	83 (73%)	65 (57%)	80 (71%)
MPA	21	18 (86%)	12 (57%)	20 (95%)	15 (71%)	20 (95%)	15 (71%)
eGPA	12	3 (25%)	2 (17%)	6 (50%)	3(25%)	5 (42%)	3 (25%)
UndifferentiatedAAV	39	37 (95%)	32 (82%)	38 (97%)	38 (97%)	39 (100%)	36 (93%)

**The specific ANCA Associated Vasculitides samples tested are outlined in the table below

17.1 Clinical sensitivity and specificity

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

Additionally, 287 clinically or analytically characterized serum samples were tested at Inova (Site#1) and at two external clinical sites (Site#2 and Site #3) with both Ethanol and Formalin ANCA, as described in section "Between sites/instruments reproducibility". The contained 49 analytically characterized samples that were excluded from the sensitivity and specificity calculations (clinical cohort n=238). The composition of the combined clinical cohort (n=653) is described in the "Clinical Performance" section.

Sensitivity (on ANCA-associated vasculitis – AAV) and specificity, calculated on the combined population, are shown below.

Ethanol:

Digital:

	cANCA Pattern		pANCA Pattern
	Sensitivity (%)(95% CI)	Specificity (%)(95% CI)	Sensitivity (%)(95% CI)	Specificity (%)(95% CI)
GPA	29.2 (21.6-38.23)	91.6 (88.0-94.1)	27.4 (20.1-36.3)	66.6 (61.2-71.5)
MPA	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	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	24.0 (17.8-31.5)	91.6 (88.0-94.1)	37.0 (29.6-45.1)	66.6 (61.2-71.5)

{42}------------------------------------------------

Manual:

	cANCA Pattern		pANCA Pattern
	Sensitivity (%)(95% CI)	Specificity (%)(95% CI)	Sensitivity (%)(95% CI)	Specificity (%)(95% CI)
GPA	29.2 (21.6-38.23)	91.3 (87.6-93.9)	30.1 (22.4-39.1)	59.7 (54.2-64.9)
MPA	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	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	24.0 (17.8-31.5)	91.3 (87.6-93.9)	39.0 (31.5-47.1)	59.7 (54.2-64.9)

NOVA View:

	cANCA Pattern		pANCA Pattern
	Sensitivity (%)(95% CI)	Specificity (%)(95% CI)	Sensitivity (%)(95% CI)	Specificity (%)(95% CI)
GPA	12.4 (7.2-20.4)	95.9 (93.0-97.7)	25.8 (18.1-35.3)	69.6 (64.1-74.6)
MPA	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	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	9.3 (5.4-15.6)	95.9 (93.0-97.7)	9.3 (5.4-15.6)	69.6 (64.1-74.6)

Formalin (C-ANCA):

Digital:

	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)

Manual:

	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)

NOVA View:

	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)

{43}------------------------------------------------

Sensitivity and specificity were also calculated on the 238 clinical samples that were run during the between sites reproducibility study at all three sites (patterns were not taken into consideration when calculating sensitivity and specificity in this section):

Site #1 (Inova), Ethanol ANCA

n=238	Sensitivity (%)(95% CI)	Specificity (%)(95% CI)
Manual	88.8 (80.0-94.0)	50.6 (42.9-58.3)
Digital	87.5 (78.5-93.1)	57.6 (49.8-65.0)
NOVA View	85.0 (75.6-91.2)	62.7 (54.9-69.8)

Site #1 (Inova), Formalin ANCA

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

Site #2, Ethanol ANCA

n=238	Sensitivity (%) (95% CI)	Specificity (%) (95% CI)
Manual results	75.0 (64.5-83.2)	57.0 (49.2-64.4)
Digital results	71.3 (60.5-80.0)	65.6 (57.9-72.6)
NOVA View results	70.0 (59.2-78.9)	69.5 (61.8-76.2)

Site #2, Formalin ANCA

n=238	Sensitivity (%)(95% CI)	Specificity (%)(95% CI)
Manual	80.0 (70.0-87.3)	90.5 (84.9-94.2)
Digital	81.3 (71.3-88.3)	94.3 (89.5-97.0)
NOVA View	76.3 (65.9-84.2)	94.3 (89.5-97.0)

Site #3, Ethanol ANCA

n=238	Sensitivity (%)(95% CI)	Specificity (%)(95% CI)
	Manual	72.5 (61.9-81.1)
Digital	72.5 (61.9-81.1)	65.8 (58.1-72.8)
NOVA View	70.0 (59.2-78.9)	68.4 (60.7-75.1)

{44}------------------------------------------------

Site #3, Formalin ANCA

n=238	Sensitivity (%)(95% CI)	Specificity (%)(95% CI)
Manual	77.5 (67.2-85.3)	93.0 (88.0-96.1)
Digital	76.3 (65.9-84.2)	94.3 (89.5-97.0)
NOVA View	75.0 (64.5-83.2)	96.8 (92.8-98.6)

The reason for the low specificity of Ethanol ANCA testing is assumed to be the consequence of ANA interference/cross-reactivity, as many of the control groups (rheumatoid arthritis, thyroid disease, celiac disease, etc.) are known to have an ANA prevalence above that of the healthy population. As ANCA is tested in 1:20 serum dilution, this effect is augmented. However, it is also known that ANA becomes negative, or shows a highly decreased staining intensity on Formalin fixed ANCA slides. Therefore the relationship between Ethanol and Formalin ANCA results was assessed on the control population of the between sites reproducibility study (n=158).

The vast majority of Ethanol ANCA positive samples became negative on Formalin ANCA, as shown in the Tables below (only Inova data are shown):

	NOVA View Result Formalin
NOVA View Result Ethanol	neg	pos	Total
neg	99	0	99
pos	54	5	59
Total	153	5	158

	Manual Result Formalin
Manual Result Ethanol	neg	pos	Total
neg	78	2	80
pos	65	13	78
Total	143	15	158

	Digital Results, Formalin
Digital Results Ethanol	neg	pos	Total
neg	90	1	91
pos	56	11	67
Total	146	12	158

It was confirmed that the remaining Formalin positive samples showed significant decrease in staining intensity, implying that those were ANA positive samples:

	Median LIU,NOVA View	Median grade,manual	Median grade,digital
Ethanol ANCA	553	2.5	2.5
Formalin ANCA	76	1.0	1.0

{45}------------------------------------------------

17.2 Expected values

Expected values were analyzed on 89 samples from apparently healthy subjects: 29 females, 6 with unknown gender, with mean age of 45.7 years (range of 18-68).

With Ethanol ANCA, there were 4 positive results with NOVA View software interpretation, 8 with manual interpretation, and 4 with digital interpretation.

There were no positive results with Formalin ANCA testing, by any interpretation method.

18. System description

18.1 NOVA View Automated Fluorescence Microscope and Software

18.1.1 NOVA View Automated Fluorescence Microscope

NOVA View® Automated Fluorescence Microscope is an automated system consisting of a fluorescence microscope and software that acquires, analyzes, stores and displays digital images of stained indirect immunofluorescent slides. It is intended as an aid in the detection and classification of certain antibodies by indirect immunofluorescence technology. The device can only be used with cleared or approved in vitro diagnostic assays that are indicated for use with the device. A trained operator must confirm results generated with the device.

The NOVA View device has been cleared by the FDA in DEN140039. Subsequently, the use of NOVA View with AUTOLoader has been cleared in K153150.

Device description and Principle of Operation were described in DEN140039 and K153150, and remained unchanged for this submission.

18.1.2 Software

Level of Concern: Moderate.

The NOVA View software version in this submission contains the following changes compared to the version in K153150:

Addition of ANCA module, containing ANCA Ethanol and ANCA Formalin applications, and ANCA SWT application.

When ANCA slides are analyzed by NOVA View, digital images of representative fields of view of the well are captured. At the same time when digital images are taken, NOVA View measures the FITC light intensity of the cells that are included in the region. NOVA View reports the measured fluorescence intensity in units of Light Intensity Units (LIU).

NOVA View provides the trained operator with the acquired digital images and the following supportive information:

LIU value

{46}------------------------------------------------

Negative/positive classification
Pattern information

Patterns reported by NOVA View (Ethanol ANCA)
P	P-ANCA
C	C-ANCA
U	Unrecognized

Patterns reported by NOVA View (Formalin ANCA)
N	Nuclear
C	Cytoplasmic
U	Unrecognized

Additionally, five programmable pattern buttons are available on the user interface that can be customized based on the end user lab's preferences and routine nomenclature.

NOVA View Ethanol ANCA Single Well Titer (SWT)

The SWT is a software 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. The highest titer that can be differentiated is 1:1280 for both P-ANCA and C-ANCA. Above this value the NOVA View reports ≥ 1280 titer.

The SWT function was established on 10 anti-MPO (P-ANCA) and 10 anti-PR3 (C-ANCA) positive samples, containing various levels of antibodies. Two-fold serial dilutions were made from each sample, starting from 1:20, up to 1:10,240. Each dilution was processed on ANCA Ethanol slides, and the results were interpreted by NOVA View and by manual reading. Based on the results that were generated on these samples, P-ANCA and C-ANCA specific LIU curves were established based on 4-PL logistic curve fitting. NOVA View uses these built-in curves for titer determination.

The validation of the SWT function was performed on altogether 24 anti-MPO (P-ANCA) and 23 anti-PR3 (C-ANCA) positive samples. All samples were serially diluted and manually titrated, and also run on the NOVA View. SWT was determined for all samples.

Acceptance criteria:

. SWT is within ± 2 dilution steps of that of the manual titer and the digital titer.
Based on 47 samples, 76.6% of SWT results were within ± 1 dilution step of that of the manual titer, and 91.5% were within ± 1 dilution step of that of the digital titer, and 97.7% of SWT results were within ± 2 dilution steps of that of both the manual titer and digital titer. One out of the 47 samples was outside of this range.

{47}------------------------------------------------

SWT, n=47	within ± 1dilution step	within ± 2dilution steps
Manual endpoint	76.6%	97.9%
Digital endpoint	91.5%	97.9%

Additionally, SWT validation was part of the between sites reproducibility study.

Seven positive samples were assayed by all three testing sites (including Inova). 85.7% (12 out of 14) of SWT results at the two external sites were within ± 1 dilution step of that of the manual titer, and 92.9% (13 out of 14) were within ± 1 dilution step of that of the digital titer; 100% of SWT results were within ± 2 dilution steps of that of both the manual titer and digital titer.

Regulation Number and Section

§ 866.5660 Multiple autoantibodies immunological test system.

(a)
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).(b)
Classification. Class II (performance standards).

Precision Performance:

Interference:

Cross-reactivity:

Conjugate Comparison (to predicate device):

Clinical Sensitivity and Specificity:

Leonthena R. Carrington -S

Indications for Use

Device Name

Indications for Use (Describe)

CONTINUE ON A SEPARATE PAGE IF NEEDED.

DO NOT SEND YOUR COMPLETED FORM TO THE PRA STAFF EMAIL ADDRESS BELOW.

NOVA Lite® DAPI ANCA (Ethanol) Kit, NOVA Lite® DAPI ANCA (Formalin) Kit

510(k) Summary

Table of Contents

1. Sponsor

2. Purpose of submission

3. Devices in the submission

4. Primary contact

5. Secondary contact

6. Product Information and Classification

6.1 Proprietary and established names

6.2 Regulatory information

7. Intended Use

8. Indications for Use

9. Predicate device

10. Comparison matrix to predicate device

11. Type of the product

12. Device description

13. Principle of the method and summary of the procedure

Manual interpretation

Ethanol ANCA:

Formalin ANCA:

NOVA View interpretation

14. Analytical performance characteristics

14.1 Nomenclature used in the studies

14.2 Precision performance

14.2.1 Within laboratory imprecision

NOVA Lite® DAPI ANCA Ethanol and Formalin

Page 11 of 45

ANCA Formalin repeatability and reproducibility

14.2.2 Between lots reproducibility

Qualitative Agreement, Ethanol ANCA:

NOVA View Summary Table

Manual Summary Table

Digital Summary Table

Qualitative Agreement, Formalin ANCA:

NOVA View Summary Table

Manual Summary Table

Digital Summary Table

Grade agreement, Ethanol ANCA:

Grade agreement, Formalin ANCA:

Manual grade comparison:

Digital grade comparison:

within± one grade:

Pattern agreement, Ethanol ANCA:

Pattern agreement, Formalin ANCA:

14.2.3 Between sites/instruments reproducibility

Site #1 (Inova)

Site #2

Site #3

Formalin ANCA

Ethanol ANCA

Pattern agreement:

Qualitative agreement:

Pattern agreement:

14.2.4 Between operators reproducibility

Ethanol ANCA, NOVA View Results

Summary of SD and %CV:

Ethanol ANCA, manual reading Qualitative agreement:

Grade agreement:

Ethanol ANCA, digital reading Qualitative agreement:

Grade agreement:

Ethanol ANCA

Formalin ANCA, NOVA View Results

Summary of SD and %CV:

Grade agreement:

Formalin ANCA, digital reading Qualitative agreement:

Grade agreement:

Formalin ANCA

14.3 Linearity and Analytical Measuring Range (AMR)