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NOVA Lite® DAPI dsDNA Crithidia luciliae is an indirect immunofluorescent assay for the qualitative and/or semi-quantitative determination of anti-double stranded DNA (dsDNA) IgG antibodies in human serum by NOVA View Automated Fluorescence Microscope or manual fluorescence microscopy. The presence of anti-dsDNA can be used in conjunction with other serological and clinical findings to aid in the diagnosis of systemic lupus erythematosus (SLE). All results generated with NOVA View device must be confirmed by a trained operator.

The NOVA Lite DAPI dsDNA Crithidia luciliae Kit is an indirect immunofluorescence assay for the qualitative detection and semi-quantitative determination of Anti-dsDNA Antibodies (IgG) in human serum. Samples are diluted 1:10 in PBS and incubated with the antigen substrate (dsDNA on glass microscope slides). After incubation, unbound antibodies are washed off. The substrate is then incubated with antihuman IgG-FITC conjugate. The conjugate contains a DNA-binding blue fluorescent dye, 4',6-diamidino-2phenylindole (DAPI) that is required for NOVA View use. The blue dye is not visible by traditional fluorescence microscope at the wavelength where FITC fluorescence is viewed. Unbound reagent is washed off. Stained slides are read by manual fluorescence microscope or scanned with the NOVA View Automated Fluorescence Microscope. The resulting digital images are reviewed and interpreted from the computer monitor. dsDNA positive samples exhibit an apple green fluorescence corresponding to areas of the substrate where autoantibody has bound.

Here's a breakdown of the acceptance criteria and study details for the NOVA Lite DAPI dsDNA Crithidia luciliae Kit, based on the provided text:

Acceptance Criteria and Reported Device Performance

Study Details:

1. Sample sizes used for the test set and the data provenance:

   • Precision Study: 6 samples (2 negative, 2 borderline, 2 positive), each processed in triplicate across 14 runs (2 runs/day for 7 days), resulting in 42 data points per sample.
   • Reproducibility Studies (Between sites/instruments): 10 samples (3 negative, 7 positive), each tested in triplicate, twice a day for 5 days at each of 3 sites. This results in 30 data points per sample per site, or 90 data points per sample across all sites. Total data points for this study: 10 samples * 30 data points/sample * 3 sites = 900 data points.
   • Reproducibility (Between lots): 20 clinically and/or analytically characterized samples, tested in duplicate.
   • Linearity Study: 3 positive samples (high, medium, low), serially diluted from 1:10 up to 1:5120. (Number of replicates not specified for this part, but results are given for each dilution).
   • Interference Study: 3 specimens (one negative, one positive, one strong positive) for each interferent, with interfering substances spiked at three different concentrations in 10% of total specimen volume. Samples assessed in triplicates.
   • Sample Stability and Handling: 3 samples (negative, cut-off, positive), tested in duplicates for various conditions (up to 21 days at 2-8°C, up to 48 hours at room temperature, up to 3 freeze/thaw cycles).
   • Reagent Stability (Shelf-life): 3 lots of the kit, tested over 4 weeks accelerated stability (each week = 6 months real time). Real-time stability data was available up to 24, 15, and 19 months for the respective lots at the time of submission.
   • Reagent Stability (In-use/Open Vial): Not detailed how many units/tests were performed each week for 8 weeks.
   • Clinical Performance (Initial Study): 766 clinically characterized serum samples (391 SLE, 375 other diseases). No explicit country of origin is stated, but given this is an FDA submission for Inova Diagnostics, Inc. in San Diego, California, it is reasonable to infer a US-centric data provenance. The study appears to be retrospective based on "clinically characterized serum samples."
   • Clinical Performance (3 Sites Study): 269 clinically characterized samples tested at three sites. Total for reporting: 100 positive (SLE) and 169 negative (non-SLE) per site. The samples comprise 300 SLE and 507 non-SLE clinical diagnoses in total across the three sites. The data provenance is likely multi-center, potentially within the US. The description "clinically characterized samples" suggests these were collected and diagnosed prior to the study, implying a retrospective nature.
   • Expected Values: 120 samples from apparently healthy subjects (60 females, mean age 41, range 18-73).
   • Comparison with Predicate Device: The same 744 serum samples used in the initial clinical study (391 SLE, 353 other diseases).
   • SWT Validation: 31 positive samples for initial validation. 7 positive samples in the between-sites reproducibility study.

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

   • For clinical studies: The ground truth for the clinical studies is stated as "clinically characterized serum samples" and "clinical diagnosis." This implies that the samples were obtained from patients with established diagnoses, likely made by medical professionals (e.g., rheumatologists for SLE patients). The text does not specify the number of experts or their exact qualifications (e.g., "Radiologist with 10 years of experience" is not mentioned, as this is an immunoassay, but rather "clinicians" or "diagnosticians").
   • For analytical studies (Precision, Reproducibility, Linearity, Interference, Stability): The ground truth (Expected Result/Expected Grade) for the control samples or known samples was established by the manufacturer, often based on previous characterization or established laboratory practices. The interpretation of "Manual Reading" and "Digital Reading" results are performed by "trained operators."

3. Adjudication method for the test set:

   • For analytical results (Precision, Reproducibility, Linearity, Interference, Stability): The text mentions that "Digital images were interpreted and confirmed" in multiple sections (e.g., Linearity, Interference, Sample Stability). For the "Reproducibility Studies (Between sites/instruments)", manual and digital reading was performed by "two operators at each site, to assess between operator reproducibility." The acceptance criteria then focus on agreement percentages between operators. This implies that if disagreements occurred, they were likely adjudicated to reach the "Summary" percentages. However, a specific formal adjudication method like "2+1" or "3+1" is not explicitly stated.
   • For clinical results: The clinical samples were "clinically characterized," meaning their diagnosis served as the ground truth. There's no indication of an adjudication process for these clinical diagnoses within the context of this device study.

4. 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:

   • This is not a traditional MRMC comparative effectiveness study involving AI assistance improving human readers. The study compares three modes of interpretation:
     • Manual Reading: Human interpretation using a traditional fluorescence microscope.
     • Digital Reading: Human interpretation of NOVA View generated images on a computer monitor.
     • NOVA View (Software): Automated interpretation by the device's software (algorithm only), which is then confirmed by a trained operator.
   • Therefore, the setup is more of a comparison between manual microscopy, human interpretation of digital images, and the device's automated output. The device itself (NOVA View) is not presented as an AI-assistance tool for human readers but as an alternative interpretation method that still requires human confirmation.
   • The "effect size of how much human readers improve with AI vs without AI assistance" is not directly measured in this context because the "NOVA View" results are the algorithm's output, not a human reader assisted by the algorithm. The "Digital Reading" is human interpretation of the images produced by the NOVA View device, which might be considered an "assisted" or "different modality" reading but not in the typical AI-driven improvement sense.

   Let's look at sensitivity/specificity to show the comparison between manual, digital (human on digital images), and NOVA View (algorithm):

   Initial Clinical Study (N=766)

   • Sensitivity (on SLE):
     • Manual: 48.1% (43.2-53.0)
     • Digital: 48.1% (43.2-53.0)
     • NOVA View: 57.0% (52.1-61.8)
   • Specificity:
     • Manual: 91.2% (87.9-93.7)
     • Digital: 92.3% (89.1-94.6)
     • NOVA View: 88.8% (85.2-91.6)

   Clinical Studies 3 Sites (N=807)

   • Sensitivity (on SLE):
     • Manual Reading: 32.7% (27.6-38.2)
     • Digital Reading: 34.0% (28.9-39.5)
     • NOVA View: 40.0% (34.6-45.6)
   • Specificity:
     • Manual Reading: 95.5% (93.3-97.0)
     • Digital Reading: 95.5% (93.3-97.0)
     • NOVA View: 85.4% (82.1-88.2)

   In both clinical studies, the NOVA View algorithm demonstrates higher sensitivity for SLE detection compared to manual or digital human readings, but lower specificity. This highlights a performance difference, not an improvement of human readers with AI assistance.

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

   • Yes, the "NOVA View" performance reported in the tables (e.g., sensitivity, specificity, qualitative agreements in reproducibility studies) represents the standalone algorithm's performance.
   • The text explicitly states: "All results generated with NOVA View device must be confirmed by a trained operator." This indicates that while the software generates automated classifications, the final clinical interpretation includes a human-in-the-loop for confirmation. The reported performance metrics for "NOVA View" specifically reflect the device's automated output.

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

   • Clinical Ground Truth: "Clinical diagnosis" for patient-derived samples (e.g., Systemic Lupus Erythematosus (SLE), Drug Induced Lupus, Infectious Disease, etc.). This is likely based on a combination of clinical findings and serological tests by clinicians. It is stated as "clinically characterized serum samples."
   • Analytical Ground Truth: For the precision, reproducibility, linearity, interference, and stability studies, the "Expected Result" or "Expected Grade" for the tested samples serves as the ground truth. These are typically reference materials or well-characterized samples with known positive/negative status or reactivity grades established by the manufacturer.

7. The sample size for the training set:

   • The document does not explicitly state the sample size of a training set for the NOVA View algorithm. It describes validation studies (test sets) for the kit and the performance of the NOVA View device, but not how the algorithm itself was developed or trained.
   • What is mentioned is that for the SWT (Single Well Titer) feature, "The SWT function was established using 22 dsDNA positive samples that represent various levels of antibodies." However, this refers to establishing the intensity curves for titer determination, not necessarily a broad 'training set' for the overall positive/negative classification logic or image analysis.

8. How the ground truth for the training set was established:

   • As the training set size is not stated, neither is the method for establishing its ground truth.
   • For the 22 dsDNA positive samples used to establish SWT intensity curves, it is implied that manual and digital readings (human interpretations) served as comparison points for establishing the LIU (Light Intensity Units) to titer relationship. The validation of SWT compares its output to manual and digital end-point titers.

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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.

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

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

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.

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NOVA Lite® DAPI ANA Kit is an indirect immunofluorescence assay for the qualitative detection and semi-quantitative determination of anti-nuclear antibodies of the IgG isotype in human serum by manual fluorescence microscopy or with the NOVA View Automated Fluorescence Microscope. The presence of anti-nuclear antibodies can be used in conjunction with other serological tests and clinical findings to aid in the diagnosis of systemic lupus erythematosus and other systemic rheumatic diseases. A trained operator must confirm results when generated with the NOVA View device.

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

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

The provided document describes the analytical and clinical performance of the NOVA Lite® DAPI ANA Kit, an indirect immunofluorescence assay for detecting anti-nuclear antibodies. The study focuses on demonstrating substantial equivalence to a predicate device and the agreement between manual microscopy, digital image interpretation, and the automated NOVA View system.

Here's a breakdown of the requested information:

1. Table of Acceptance Criteria and Reported Device Performance

• Pattern Agreement (Clinical Cohort): Agreement between digital image reading and manual reading was above 90% at all three sites (94.7% Site 1, 91.6% Site 2, 95.7% Site 3). |
  | Clinical Sensitivity & Specificity | Sensitivity and specificity values at each site should have overlapping confidence intervals between NOVA View classification, digital image reading, and manual reading, indicating no significant differences. | - Site 1: Overlap observed (e.g., SLE sensitivity for NV: 80.0%, Manual: 72.0%, Digital: 80.0%; SARD+AIL sensitivity for NV: 69.4%, Manual: 62.9%, Digital: 69.9%; Specificity for NV: 75.3%, Manual: 74.1%, Digital: 72.4%).- Site 2: Overlap observed (e.g., SLE sensitivity for NV: 72.0%, Manual: 70.7%, Digital: 73.3%; SARD+AIL sensitivity for NV: 62.9%, Manual: 65.6%, Digital: 62.98%; Specificity for NV: 77.0%, Manual: 67.2%, Digital: 75.3%).- Site 3: Overlap observed (e.g., SLE sensitivity for NV: 82.7%, Manual: 82.7%, Digital: 81.3%; SARD+AIL sensitivity for NV: 72.0%, Manual: 71.0%, Digital: 69.4%; Specificity for NV: 69.0%, Manual: 67.2%, Digital: 71.3%).- No statistically significant differences were found between the different reading methods. |
  | CDC ANA Reference Sera | All reference sera should produce the expected pattern. Results of NOVA View digital image interpretation should be within ± one reactivity grade from manual interpretation. No discrepancies in pattern interpretation. | - All reference sera produced the expected pattern.- Digital image interpretation results were within ± one reactivity grade from manual interpretation.- No discrepancies in pattern interpretation were seen between manual and digital results. |

2. Sample Sizes Used for the Test Set and Data Provenance

• Precision/Reproducibility Studies:
  • First Set: 13 samples (3 negative, 10 positive), processed in 3 replicates across 10 runs (30 data points per sample).
  • Second Set: 22 samples (20 negative/around cut-off, 2 strong positive), processed in 3 replicates across 10 runs (30 data points per sample).
  • Third Set: Samples tested in triplicates or duplicates across 5 runs (15 or 10 data points per sample).
• Conjugate Comparison: 407 individual human serum samples.
• Method Comparison: 410 samples (400 clinically characterized sera, 10 samples with known ANA patterns).
• Lot-to-Lot Comparison: 40 sera.
• Endpoint Titration Accuracy: 10 ANA positive samples.
• Agreement on Clinical Sample Cohort (Reproducibility): 120 samples at each of 3 sites.
• Clinical Performance (Clinical Sensitivity and Specificity): 463 clinically characterized samples at each of 3 sites.
• CDC ANA Reference Sera: 12 reference sera.

Data Provenance:

• The document implies that the studies were conducted by Inova Diagnostics (Site #1) and two external sites (Site #2 and Site #3). While origin of patients' samples (e.g., country) is not explicitly stated for all cohorts, the studies conducted at "external sites" suggest broader geographic reach for sample collection, and certainly implies varied patient populations.
• The studies were retrospective, using "clinically characterized samples" and "individual serum samples" that were already available.

3. Number of Experts Used to Establish the Ground Truth for the Test Set and Their Qualifications

The document consistently states that "A trained operator must confirm results when generated with the NOVA View device" and that "all slides were read by the same operator with manual microscopy" for various studies. For adjudication specifically, the number of experts for initial ground truth establishment isn't broken down for each individual sample, but implies internal validation by "trained operators".

• Precision Studies, Conjugate Comparison, Accelerated Stability, CDC Reference Sera: "The slides were read with NOVA View, and digital images were interpreted by the operator." and "all slides were read by the same operator with manual microscopy." This suggests at least one trained operator for defining ground truth for reading discrepancies.
• Endpoint Titration and Reproducibility/Clinical Performance Studies: "all slides were read by the same operator with manual microscopy." for generating ground truth. These studies were carried out at three different sites (Inova Diagnostics and two external locations), implying that a "trained operator" at each site was responsible for manual readings. The qualifications of these "trained operators" are not further specified beyond "trained operator."

4. Adjudication Method for the Test Set

The primary method for establishing agreement and performance comparison appears to be through comparison with manual microscopy readings by a "trained operator". There is no explicit mention of an adjudication protocol (e.g., 2+1 or 3+1 consensus) for discrepant results between the automated system and manual reading, or between multiple manual readers for the general test sets. The "trained operator" performing the manual reading effectively serves as the reference standard against which the digital and NOVA View results are compared. For the NOVA View results, it states "Digital images were interpreted and confirmed," implying a human review step and potential individual reconciliation.

5. If a Multi-Reader Multi-Case (MRMC) Comparative Effectiveness Study Was Done, and the Effect Size of How Much Human Readers Improve with AI vs. Without AI Assistance

• No explicit MRMC comparative effectiveness study involving AI-assistance performance improvement for human readers is described. The studies primarily focus on the agreement and equivalence between:

  1. Manual reading (human only, traditional method)
  2. Digital image reading (human interpreting images from the automated system)
  3. NOVA View output (raw automated classification)

  The design compares the performance of the automated system and its digital interpretation against the manual method, rather than quantifying how much human readers improve when assisted by the AI in making their initial assessments. The phrasing "A trained operator must confirm results when generated with the NOVA View device" suggests that the human remains in the loop for final confirmation, but the study doesn't isolate the "effect size" of this assistance.

6. If a Standalone (i.e., algorithm only without human-in-the-loop performance) Was Done

Yes, partial standalone performance data is presented as "NOVA View output".
"NOVA View" refers to "raw results obtained with the NOVA View Automated Fluorescence Microscope, such as Light Intensity Units (LIU), positive/negative classification and pattern information." These raw results are then compared against "Digital reading" (human interpretation of NOVA View images) and "Manual reading" (human interpretation of actual slides).

For example, in the "Precision performance" section, NOVA View output (standalone) is compared to digital image reading. In the "Clinical performance" section, NOVA View classification (standalone) is compared to digital image reading and manual reading for sensitivity, specificity, and agreement.

7. The Type of Ground Truth Used

The primary ground truth used for performance validation is expert consensus/manual interpretation by experienced "trained operators" using traditional fluorescence microscopy. This is explicitly stated in multiple sections, for instance: "all slides were read by the same operator with manual microscopy" serving as a reference.

For the clinical performance section, "clinically characterized samples" are used, implying that patient diagnoses (e.g., SLE, SSc, SS, etc.) served as the clinical classification for sensitivity/specificity calculations, but the ANA ground truth itself (positive/negative, pattern, grade) within those clinical cohorts was established by manual interpretation.

The CDC ANA reference sera also represent a form of "known ground truth" based on established reference standards and known antibody specificities.

8. The Sample Size for the Training Set

The document does not explicitly state the sample size of the training set used to develop or train the NOVA View automated system's algorithms. The focus of this 510(k) submission is on the validation of the NOVA Lite® DAPI ANA Kit, which includes its use with the previously cleared NOVA View device (DEN140039). Training data details for NOVA View itself would likely be in its original submission.

9. How the Ground Truth for the Training Set Was Established

Since the document does not specify the training set used for the NOVA View algorithm, it also does not describe how its ground truth was established. This information would typically be found in the original submission for the NOVA View device itself (DEN140039).

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