ImmuGlo HEp-2 Elite IFA

K172745 · Immco Diagnostics, Inc. · DHN · Jun 5, 2018 · Immunology

Device Facts

• Record ID: K172745
• Device Name: ImmuGlo HEp-2 Elite IFA
• Applicant: Immco Diagnostics, Inc.
• Product Code: DHN · Immunology
• Decision Date: Jun 5, 2018
• Decision: SESE
• Submission Type: Traditional
• Regulation: 21 CFR 866.5100
• Device Class: Class 2

Indications for Use

The ImmuGlo HEp-2 Elite IFA is an indirect immunofluorescence antibody test for the qualitative or semi-quantitative detection of anti-nuclear antibodies (ANA) of the IgG isotype in human serum utilizing standard HEp-2 cells and engineered HEp-2 cells as a substrate. The ImmuGlo HEp-2 Elite IFA is intended for use as an aid in the diagnosis of systemic rheumatic diseases in conjunction with other clinical and laboratory findings.

Device Story

Indirect immunofluorescence assay (IFA) kit; detects IgG anti-nuclear antibodies (ANA) in human serum. Substrate consists of barcoded glass slides with monolayer of standard HEp-2 cells and engineered PSIP1-knockout HEp-2 cells (1:9 ratio). Patient serum incubated on slides; bound antibodies detected via fluorescein-labeled anti-human IgG conjugate with Evan's blue counterstain. Manual operation by laboratory personnel using fluorescence microscope (200x+ magnification, 490nm excitation/510nm barrier filters). Engineered cells lack LEDGF antigen, aiding discrimination of DFS70 pattern from homogeneous/speckled patterns. Output is qualitative (positive/negative) or semi-quantitative (titer) based on fluorescence intensity and pattern recognition. Results aid clinicians in diagnosing systemic rheumatic diseases (SARD) alongside other clinical/laboratory findings.

Clinical Evidence

Clinical performance was evaluated using 256 ANA-associated disease samples and 497 autoimmune/infectious disease controls. Sensitivity for ANA-associated diseases was 78.1% (95% CI: 72.5-82.9%) and specificity was 75.1% (95% CI: 71.0-78.7%). A pattern agreement study with 243 samples across 3 blinded readers showed high concordance for DFS70, homogeneous, and speckled patterns. Precision studies (repeatability and reproducibility) across multiple sites and operators met acceptance criteria for qualitative, titer, and fluorescence intensity agreement. No significant interference was observed with common medications or endogenous substances.

Technological Characteristics

Indirect immunofluorescence assay. Substrate: barcoded glass slides with standard and PSIP1-knockout HEp-2 cells. Reagents: FITC-conjugated anti-human IgG, Evan's blue counterstain, PBS wash buffer, mounting medium. Manual fluorescence microscopy (490nm/510nm filters). Storage 2–8°C. No software/algorithm; manual visual interpretation of fluorescence patterns.

Indications for Use

Indicated for qualitative or semi-quantitative detection of IgG anti-nuclear antibodies (ANA) in human serum to aid in the diagnosis of systemic rheumatic diseases. For prescription use only.

Regulatory Classification

Identification

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

Predicate Devices

• ImmuGlo ANTINUCLEAR ANTIBODY (ANA) TEST (HEP-2) CELLS (K883883)

Submission Summary (Full Text)

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# 510(k) SUBSTANTIAL EQUIVALENCE DETERMINATION DECISION SUMMARY

A. 510(k) Number:
K172745

B. Purpose for Submission:
New device

C. Measurand:
IgG Anti-Nuclear Antibodies (ANA)

D. Type of Test:
Qualitative or semi-quantitative, indirect immunofluorescence

E. Applicant:
IMMCO Diagnostics, Inc.

F. Proprietary and Established Names:
ImmuGlo HEp-2 Elite IFA

G. Regulatory Information:

1. Regulation section:
21 CFR §866.5100 – Antinuclear antibody immunological test system

2. Classification:
Class II

3. Product code:
DHN – Antinuclear Antibody, Indirect Immunofluorescent, Antigen, Control

4. Panel:
Immunology (82)

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H. Intended Use:

1. Intended use:

The ImmuGlo HEp-2 Elite IFA is an indirect immunofluorescence antibody test for the qualitative or semi-quantitative detection of anti-nuclear antibodies (ANA) of the IgG isotype in human serum utilizing standard HEp-2 cells and engineered HEp-2 cells as a substrate. The ImmuGlo HEp-2 Elite IFA is intended for use as an aid in the diagnosis of systemic rheumatic diseases in conjunction with other clinical and laboratory findings.

2. Indication for use:

Same as Intended Use

3. Special conditions for use statement:

For Prescription Use Only

4. Special instrument requirements:

Fluorescence microscope for manual operation
200× or higher magnification, 490 nm excitation filter, 510 nm barrier filter

I. Device Description:

The kit is an indirect immunofluorescence assay (IFA) to detect IgG anti-nuclear antibodies (ANA) against proteins presented in HEp-2 cell lines. The standard test kit contains:

- Substrate slides: barcoded glass slides with standard and engineered DFS70-knock-out HEp-2 cells attached in a monolayer in the slide wells
- ANA homogeneous pattern positive control
- ANA DFS70 pattern positive control
- Negative Control
- Conjugate: anti-human IgG FITC conjugate containing Evan's blue counterstain. Binds fluorescein to reactive antibodies reacting against HEp-2 antigens
- Buffered diluent provided for specimen dilutions
- Wash buffer: phosphate buffered saline (PBS) diluent removes reagents and unbound antibodies after incubation steps
- Mounting medium provided for coverslipping of slides to view under fluorescence microscope
- Coverslips

Optional components:

The standard kit format supplies the Conjugate and Evan's blue counterstain as one pre-formulated reagent, however, they may also be supplied separately to accommodate laboratory workflow. The formulation is intended to be separate but functionally identical.

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J. Substantial Equivalence Information:

1. Predicate device name:
ImmuGlo ANTINUCLEAR ANTIBODY (ANA) TEST (HEP-2) CELLS

2. Predicate 510(k) number:
K883883

3. Comparison with predicate:

| Similarities | | |
| --- | --- | --- |
| Item | Device | Predicate |
| Intended Use | The ImmuGlo HEp-2 Elite IFA is an indirect immunofluorescence antibody test for the qualitative or semi-quantitative detection of anti-nuclear antibodies (ANA) of the IgG isotype in human serum utilizing standard HEp-2 cells and engineered HEp-2 cells as a substrate. The ImmuGlo HEp-2 Elite IFA is intended for use as an aid in the diagnosis of systemic rheumatic diseases in conjunction with other clinical and laboratory findings. | Indirect immunofluorescence (IF) antibody test for the detection and quantitation of anti-nuclear antibodies (ANA) in human serum |
| Methodology | Indirect immunofluorescence | Same |
| Matrix | Serum | Same |
| Detection of antibodies | Anti-nuclear antibodies | Same |
| Component Set | Includes positive control, negative control, conjugate, diluent, wash buffer, mounting medium, substrate slides, coverslips | Same |
| Conjugate | FITC IgG | Same |
| Screening dilution | 1:40 | Same |
| Reading | Fluorescence microscope | Same |
| Storage | 2–8°C | Same |
| Differences | | |
| --- | --- | --- |
| Item | Device | Predicate |
| Substrate | Standard HEp-2 cells mixed with engineered HEp-2 with the PSIP1 gene knocked out in 1:9 ratio | Standard HEp-2 cells |
| Positive control | ANA homogenous, DFS70 Ab | ANA homogenous |

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K. Standard/Guidance Document Referenced (if applicable):

| Org | Standard ID | Version | Date | Title |
| --- | --- | --- | --- | --- |
| CLSI | EP05 | A3 | Sep 2014 | Evaluation of Precision Performance of Quantitative Measurement Methods |
| CLSI | EP06 | A | Apr 2003 | Evaluation of the Linearity of Quantitative Measurement Procedures |
| CLSI | EP07 | A2 | Dec 2002 | Interference Testing in Clinical Chemistry |
| CLSI | EP12 | A2 | Jan 2014 | User Protocol for Evaluation of Qualitative Test Performance |

L. Test Principle:

In the indirect IFA method used in this kit, patient sera are incubated on HEp-2 cell substrates to allow binding of antibodies. Any antibodies not bound are removed by washing with buffer. Bound antibodies of the IgG class are detected by incubation of the substrate with fluorescein-labeled anti-human IgG conjugate. Reactions are observed under a fluorescence microscope equipped with appropriate filters. The presence of nuclear and cytoplasmic patterns AC-1 to AC-28 as described by ICAP¹ is demonstrated by green fluorescence of specific structures in the cells. A majority (~90%) of the cells present on the substrate are devoid of a functional PSIP1 gene which eliminates the relevant antigenic target of the DFS70 pattern. This may aid in discrimination of ANA homogeneous and speckled patterns from the DFS pattern.

It is recommended that patient samples demonstrating specific fluorescence reactions at a dilution of 1:40 be reported as positive. Each laboratory must determine its own normal values to account for differences in microscopy systems, personnel, and training. The titers are determined by testing serial dilutions of patient samples and reported as the highest reciprocal dilution with a positive reaction.

M. Performance Characteristics:

1. Analytical performance:

All results presented below met the manufacturer's pre-determined acceptance criteria. Interpretation of Results: Results should be reported as negative, positive, or, if end-point titer has been determined, positive with titer. It is recommended that patient samples demonstrating specific fluorescence reactions at a dilution of 1:40 be reported as positive. Each laboratory must determine its own normal values to account for differences in microscopy systems, personnel, and training. Users should only read only fields that contain specific staining of the HEp-2 substrates.

¹ Chan EK, Damoiseaux J, Carballo OG, Conrad K, de Melo Cruvinel W, Francescantonio PL, Fritzler MJ, Garcia-De La Torre I, Herold M, Mimori T, Satoh M, von Mühlen CA, Andrade LE. “Report of the First International Consensus on Standardized Nomenclature of Antinuclear Antibody HEp-2 Cell Patterns 2014-2015” Front Immunol, 2015; 6:412. doi: 10.3389/fimmu.2015.00412. PMID: 2634773

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The International Consensus on Antinuclear Antibody Patterns report recommends a standardized nomenclature and reporting format for IFA patterns. Major nuclear patterns that are recommended to be reported include Homogeneous (AC-1), Speckled (AC-2, AC-4, AC-5), Centromere (AC-3), Discrete Nuclear Dots (AC-6, AC-7) and Nucleolar (AC-8, AC-9, AC-10). Major cytoplasmic patterns include Fibrillar (AC-15, AC-16, AC-17), Speckled (AC-18, AC-19, AC-20), Reticular/AMA (AC-21), Polar Golgi-like (AC-22) and Rods and rings (AC-23). Including the most commonly and mandated to be reported patterns described above, a total of 28 patterns are described in the ICAP reference.

Interpretation of DFS70 pattern on HEp-2 ELITE IFA substrate: In the ~10% baseline HEp-2 cells in each well, a dense fine speckled pattern will be observed on interphase nuclei. Chromatin-associated staining is seen on mitotic nuclei. The remaining ~90% of HEp-2 cells have the PSIP1 gene encoding the LEDGF antigen knocked out and therefore will not produce a similar pattern. If 10% of the HEp-2 cells have brighter fluorescence corresponding to DFS70 pattern and rest of the cells present additional patterns or fine speckled signal above cut-off, it indicates the presence of mixed patterns. Closer analysis of such patterns is essential to confirm if other antibody specificity is present in addition to the DFS70 pattern associated with LEDGF/PSIP1. Due to the high prevalence of AC-2 patterns in an ANA screening population and relatively low association with autoimmune rheumatic diseases, the ICAP committee recommends all clinical labs report the DFS70 pattern.

a. Precision/Reproducibility:

The sponsor modeled precision studies based on CLSI guideline EP05-A3.

Repeatability:

To assess repeatability of the ImmuGlo HEp-2 Elite IFA, 8 negative and 17 positive samples representing various patterns and positivities were tested: six replicates × two runs/day × five days × two operators = 120 replicate measures for each sample. There was ≥ 90% agreement for pattern calls for all samples. One speckled sample was reported with 93% pattern agreement.

Reproducibility:

To assess variability, 10 positive samples with various patterns and positivities and one negative sample were tested in triplicate in 10 runs (two runs/day × five days) at three different sites, resulting in 90 datapoints for each sample for each outcome. Global agreement values for pattern identity and titer (±1 dilution value) are provided in the table below:

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| Reproducibility of HEp-2 Elite IFA substrate across all three sites | | | | |
| --- | --- | --- | --- | --- |
| Sample | Pattern | Endpoint Titer | Pattern Agreement | Titer Agreement (±1 dilution) |
| 1 | DFS70 | 80 | 100% | 100% |
| 2* | Cytoplasmic | 80 | 100% | 100% |
| | Nuclear dots | 80 | 100% | 100% |
| 3 | Speckled | 80 | 100% | 100% |
| 4 | Centromere | 80 | 100% | 100% |
| 5 | Speckled | 80 | 100% | 100% |
| 6 | Homogeneous | 40 | 93% | 100% |
| 7 | DFS70 | 80 | 100% | 100% |
| 8 | Homogeneous | 160 | 100% | 93% |
| 9* | Cytoplasmic | 1280 | 100% | 100% |
| | Nuclear dots | 1280 | 100% | 100% |
| 10 | Nucleolar | 1280 | 100% | 100% |
| 11 | Negative | 0 | 100% | 100% |

* samples 2 and 9 are mixed pattern specificity, primary pattern is listed first

Within-Site Reproducibility: There was $\geq 90\%$ agreement for pattern call and titer agreement (±1 dilution) for all samples at all sites. All results showed $100\%$ agreement for all patterns and titers at sites 2 and 3. A site four samples showed $90\%$ agreement; all remaining samples showed $100\%$ agreement. These samples had homogeneous patterns.

Between-Site Reproducibility: To examine the site-to-site variability of pattern calls and endpoint titer values, the reproducibility datasets were reorganized to compare each site in a pairwise fashion. Results are provided in the table below:

| Reproducibility: Pattern and titer agreement for HEp-2 Elite IFA substrate between sites; for Titer Agreement values, a range of ±1 dilution was the acceptance criterion | | | | | | | |
| --- | --- | --- | --- | --- | --- | --- | --- |
| Sample | Pattern | Site 1 v Site 2 | | Site 1 v Site 3 | | Site 2 v Site 3 | |
| | | Pattern Agreement | Titer Agreement | Pattern Agreement | Titer Agreement | Pattern Agreement | Titer Agreement |
| 1 | DFS70 | 100% | 100% | 100% | 100% | 100% | 100% |
| 2* | Cytoplasmic | 100% | 100% | 100% | 100% | 100% | 100% |
| | Nuclear dots | 100% | 100% | 100% | 100% | 100% | 100% |
| 3 | Speckled | 100% | 100% | 100% | 100% | 100% | 100% |
| 4 | Centromere | 100% | 100% | 100% | 100% | 100% | 100% |
| 5 | Speckled | 100% | 100% | 100% | 100% | 100% | 100% |
| 6 | Homogeneous | 100% | 100% | 90% | 100% | 90% | 100% |
| 7 | DFS70 | 100% | 100% | 100% | 100% | 100% | 100% |
| 8 | Homogeneous | 100% | 90% | 100% | 90% | 100% | 100% |
| 9* | Cytoplasmic | 100% | 100% | 100% | 100% | 100% | 100% |
| | Nuclear dots | 100% | 100% | 100% | 100% | 100% | 100% |
| 10 | Nucleolar | 100% | 100% | 100% | 100% | 100% | 100% |
| 11 | Negative | 100% | 100% | 100% | 100% | 100% | 100% |

* samples 2 and 9 are mixed pattern specificity, primary pattern is listed first

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Lot-to-Lot Reproducibility: A lot-to-lot reproducibility study was performed using three reagent lots. Samples were selected to test negative to strong positive specimens representing all major patterns, including DFS70. Results of the study are presented in the table below:

| Lot-to-lot Reproducibility of Qualitative Agreement | | | |
| --- | --- | --- | --- |
| | Lot 1 vs. Lot 2 | Lot 1 vs. Lot 3 | Lot 2 vs. Lot 3 |
| Positive % Agreement (95% CI) | 100% (99.4–99.9%) | 100% (99.4–99.9%) | 100% (99.4–99.9%) |
| Negative % Agreement (95% CI) | 99% (98.7–99.7%) | 99% (98.7–99.7%) | 99% (98.7–99.7%) |
| Overall % Agreement (95% CI) | 100% (99.3–99.8%) | 100% (99.3–99.8%) | 100% (99.3–99.8%) |

Pattern identities were compared between lots. The number of positive specimens with consensus patterns for each lot are provided below. As determined by consensus of results, the specimens represented homogeneous, speckled, DFS70, centromere, nucleolar, cytoplasmic, and negative. Both definitive patterns and negative results were considered as pattern agreement. The results are presented in the table below:

| Lot-to-lot Reproducibility of Pattern Agreement | | |
| --- | --- | --- |
| Lot | n | % Pattern Agreement |
| 1 | 1497 | 99.6% |
| 2 | 1496 | 99.6% |
| 3 | 1498 | 99.6% |
| 1-2-3 | 4491 | 99.6% |

# b. Linearity/assay reportable range:

To assess linearity of the ImmuGlo HEp-2 Elite IFA, 16 strong positive samples with various patterns were diluted from 1:40 to end-point. Two samples were tested from each of the main ANA patterns, including mixed-pattern samples.

The following terminology is used to describe fluorescence intensity results:

| Fluorescence intensity interpretation | |
| --- | --- |
| Intensity | Interpretation |
| 0 | Negative. No fluorescence signal above background, or specific pattern |
| 1+ | Weak fluorescence and a distinguishable pattern |
| 2+ | Clearly distinguishable pattern with higher fluorescence signal relative to 1+ category |
| 3+ | Clearly distinguishable pattern with higher fluorescence signal relative to 2+ category |
| 4+ | Clearly distinguishable pattern with higher fluorescence signal relative to 3+ category |

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The samples showed decreasing fluorescence intensity with increased dilution depicted in table below. Patterns did not change when the samples were diluted.

| Fluorescence intensity as a function of increasing sample dilution on HEp-2 Elite IFA substrate | | | | | | | | | | |
| --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- |
| Sample | Pattern(s) | \( Dilution^{-1} \) | | | | | | | | |
| | | 40 | 80 | 160 | 320 | 640 | 1280 | 2560 | 5120 | 10240+ |
| 1 | Speckled | 4+ | 4+ | 3+ | 3+ | 2+ | 2+ | 1+ | 1+ | 0 |
| 2 | Speckled | 4+ | 4+ | 3+ | 3+ | 2+ | 1+ | 0 | 0 | 0 |
| 3 | Homogenous | 4+ | 4+ | 3+ | 2+ | 2+ | 1+ | 1+ | 1+ | 1+ |
| 4 | Homogenous | 3+ | 3+ | 2+ | 2+ | 2+ | 2+ | 1+ | 1+ | 1+ |
| 5 | Dense Fine Speckled (DFS) | 2+ | 2+ | 2+ | 2+ | 1+ | 1+ | 0 | 0 | 0 |
| 6 | DFS | 3+ | 3+ | 3+ | 3+ | 2+ | 2+ | 1+ | 1+ | 0 |
| 7 | Centromere | 3+ | 3+ | 2+ | 2+ | 1+ | 1+ | 0 | 0 | 0 |
| 8 | Centromere | 3+ | 3+ | 2+ | 1+ | 1+ | 1+ | 1+ | 0 | 0 |
| 9 | Nucleolar | 3+ | 3+ | 3+ | 2+ | 1+ | 1+ | 0 | 0 | 0 |
| 10 | Nucleolar | 3+ | 3+ | 2+ | 2+ | 1+ | 0 | 0 | 0 | 0 |
| 11 | Anti-Mitochondrial (AMA) | 4+ | 4+ | 4+ | 3+ | 3+ | 3+ | 3+ | 2+ | 2+ |
| 12 | \( 1^{st}: AMA \) | 4+ | 4+ | 3+ | 3+ | 3+ | 3+ | 2+ | 2+ | 1+ |
| | \( 2^{nd}: Nuclear Dots (ND) \) | 3+ | 3+ | 3+ | 3+ | 3+ | 3+ | 2+ | 1+ | 1+ |
| 13 | \( 1^{st}: DFS \) | 3+ | 3+ | 3+ | 3+ | 2+ | 2+ | 1+ | 1+ | 0 |
| | \( 2^{nd}: ND \) | 3+ | 3+ | 2+ | 1+ | 1+ | 1+ | 0 | 0 | 0 |
| 14 | \( 1^{st}: DFS \) | 3+ | 3+ | 2+ | 1+ | 1+ | 0 | 0 | 0 | 0 |
| | \( 2^{nd}: ND \) | 2+ | 2+ | 1+ | 1+ | 1+ | 0 | 0 | 0 | 0 |
| 15 | Nuclear Rim | 4+ | 4+ | 4+ | 4+ | 3+ | 3+ | 2+ | 2+ | 1+ |
| 16 | Nuclear Rim | 3+ | 2+ | 1+ | 1+ | 1+ | 0 | 0 | 0 | 0 |

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

Traceability: A recognized standard for anti-nuclear antibodies is not available.

Stability: The sponsor performed accelerated, real time, and open kit/reagent stability studies as part of design control to assign expiration dating to components and as part of ongoing quality control/quality assurance analysis. Accelerated and open kit studies were performed on three lots of components/reagents. Based on these studies, the sponsor established an 18-month expiration date. Accelerated stability studies supported maximum shelf life of 18 months from date of manufacture. Open kit stability studies supported a maximum opened shelf life of 18 months from date of opening.

d. Detection limit:

Not applicable

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e. Analytical specificity:

Cross-reactivity: Potentially cross-reactive disease sera were tested for ANA antibodies using the ImmuGlo HEp-2 Elite IFA, including 254 samples from non-systemic autoimmune rheumatic disease autoimmune disease conditions (ANCA-associated vasculitis, celiac disease, Crohn's disease, pernicious anemia, primary biliary cholangitis, Raynaud's syndrome, rheumatoid arthritis, autoimmune thyroiditis, and ulcerative colitis), 13 inflammatory disease conditions (atopic dermatitis, psoriasis), 57 cancer samples (breast, colorectal, ovarian, pancreatic, prostate), and 173 infectious disease samples (CMV, HBV, HCV, HIV, HSV, Lyme disease, mononucleosis, Rubella, syphilis, and toxoplasmosis). Positive prevalence in these cohorts was as expected in literature and comparable to the predicate device. See section M.3, below for the results of these studies.

Interference: The sponsor modeled interference studies based on CLSI EP07-A2. Interference was studied by mixing sera with known ANA antibody levels with potentially interfering substances and evaluating the deviation from expected results. No significant interference was demonstrated for the following substances at the levels indicated: hemoglobin (2 g/L), unconjugated bilirubin (342 μmol/L), rheumatoid factor (100 U/mL), cholesterol (13 mmol/L), triglycerides (3.7 mmol/L), naproxen (2170 μmol/L), ibuprofen (2425 μmol/L), prednisone (0.84 μmol/L), mycophenylate mofetil (14.01 μg/mL), hydroxychloroquine (388 ng/mL), cyclophosphamide (1437 μmol/L), rituximab (1.143 mg/mL) and belimumab (0.939 mg/mL).

While results using suspect specimens will generally produce the correct qualitative result, testing of grossly hemolyzed or lipemic samples is not recommended as stated in the product insert.

f. Assay cut-off:

The assay cutoff of 1:40 was established from literature. The recommended screening dilution is 1:40, at which a result with fluorescence intensity 1+ – 4+ and a recognizable pattern is reported as positive. The manufacturer suggests laboratories perform doubling dilutions to establish their own titering protocols. The titers of 1:40 and 1:80 are considered low titers, 1:160 and 1:320 are considered medium titers, and 1:640 and greater are considered high titers.

2. Comparison studies:

The sponsor modeled qualitative comparison studies based on CLSI EP12-A2.

a. Method comparison with predicate device:

The ImmuGlo HEp-2 Elite IFA was tested in comparison to the predicate HEp-2 substrate using sera from 587 patients, corresponding to the clinical performance

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cohorts (see Section M.3 below). Positive and negative percent agreement values were calculated, with 95% confidence intervals and results are presented in the table below:

| Qualitative (positive or negative) performance of HEp-2 Elite IFA versus predicate ANA IFA | | | |
| --- | --- | --- | --- |
| | Predicate | | |
| | | ANA(+) | ANA(-) |
| HEp-2 | ANA(+) | 297 | 5 |
| Elite | ANA(-) | 1 | 288 |
| | | 298 | 293 |

Positive Percent Agreement: 99.7% (98.2% - 99.9%)

Negative Percent Agreement: 98.3% (96.1% - 99.3%)

Pattern identity in the table below depicts the comparison of patterns between the HEp-2 Elite IFA and the predicate ANA IFA.

| Pattern agreement of HEp-2 Elite IFA versus predicate ANA IFA | | | | | | | | | | | | |
| --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- |
| | Predicate HEp-2 | | | | | | | | | | | |
| | | Ho | Sp | Cyt | DFS | Cent | AMA | Nucl | Esoteric* | Mixed | Neg | Total |
| HEp-2 Elite | Homogeneous | 77 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 77 |
| | Speckled | 1 | 122 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 123 |
| | Cytoplasmic | 0 | 0 | 19 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 19 |
| | DFS | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 |
| | Centromere | 0 | 0 | 0 | 0 | 19 | 0 | 0 | 0 | 0 | 0 | 19 |
| | AMA | 0 | 0 | 0 | 0 | 0 | 14 | 0 | 0 | 0 | 0 | 14 |
| | Nucleolar | 0 | 0 | 0 | 0 | 0 | 0 | 26 | 0 | 0 | 1 | 27 |
| | Exotics* | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 13 | 0 | 0 | 13 |
| | Mixed | 1 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 5 | 0 | 6 |
| | Negative | 2 | 2 | 0 | 0 | 0 | 0 | 1 | 0 | 0 | 288 | 293 |
| | Total | 81 | 124 | 19 | 0 | 19 | 14 | 27 | 13 | 5 | 289 | 591 |
| | Key | | | | | | | | | | | |
| | Ho: Homogeneous | | | | | Nucl: Nucleolar | | | | | | |
| | Sp: Speckled | | | | | Esoteric*: sum of the following patterns – nuclear dots, nuclear rim, centrosome | | | | | | |
| | DFS: Dense fine speckled | | | | | Neg: Negative | | | | | | |
| | Cent: Centromere | | | | | | | | | | | |
| | AMA: anti-mitochondrial antibodies | | | | | | | | | | | |

To evaluate performance in DFS-positive samples, a cohort of 122 DFS antibody-suspected samples was studied separately. This enriched cohort was tested on both the predicate HEp-2 device and the HEp-2 ELITE IFA system. The Sponsor stated that 32 of these samples were reported on the predicate to be difficult to positively distinguish DFS from homogeneous or speckled patterns and described as 'Homogeneous/Speckled/DFS unable to be discriminated'.

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Results are summarized below:

| Pattern agreement for “DFS70 suspected” samples | | | | |
| --- | --- | --- | --- | --- |
| | Predicate | | | |
| | | DFS(+) | | DFS(-)* |
| HEp-2 | DFS70(+) | 89 | 32 | 121 |
| Elite | DFS70(-) | 0 | 0 | 0 |
| | | 89 | 32 | 121 |

*includes ‘Homogeneous/Speckled/DFS unable to be discriminated’
PPA: 100% (94.8–100%)
NPA: 0% (0–13.3%)

To further evaluate the ability of the HEp-2 Elite IFA substrate to successfully determine the DFS pattern, a reader study was performed. A total of 243 samples were selected to represent ANA patterns and negative samples, with particular emphasis on DFS, as well as homogeneous and speckled, as discrimination of these two patterns is most confounded with the DFS pattern. Samples were randomized, then tested on both the HEp-2 Elite IFA and predicate, and each substrate read by three independent readers. All readers were blinded to the expected pattern and to each others' pattern calls.

Pattern identity calls were compared between the HEp-2 Elite IFA and the predicate device for each individual reader. In this analysis, a pattern call was considered agreement if the HEp-2 Elite IFA device reported the same pattern as the predicate. In addition, aHEp-2 Elite IFA to predicate comparison was made for consensus reads, in which at least any two-of-three readers agree on the identity of a pattern call. Results are presented in the table below:

| Positive and negative pattern agreement on HEp-2 Elite IFA substrate, compared to predicate for individual readers (Readers 1–3), and for consensus read of 2/3 readers. n values depict samples positive or negative by the predicate device. | | | | | | | | | |
| --- | --- | --- | --- | --- | --- | --- | --- | --- | --- |
| Pattern | | HEp-2 Elite IFA v Predicate ANA IFA | | | | | | | |
| | | Reader 1 | | Reader 2 | | Reader 3 | | Consensus (≥2/3 Readers) | |
| | | n | % (95% CI) | n | % (95% CI) | n | % (95% CI) | n | % (95% CI) |
| All* | PPA | 176 | 90.9% (85.7–94.3%) | 170 | 81.2% (74.6–86.3%) | 207 | 90.8% (86.1–94.0%) | 177 | 87.0% (81.3–91.2%) |
| | NPA | 85 | 77.6% (67.7–85.2%) | 107 | 62.6% (53.2–71.2%) | 65 | 55.4% (43.3–66.8%) | 92 | 71.7% (61.8–79.9%) |
| DFS70** | PPA | 26 | 100% (87.1–100%) | 21 | 95.2% (77.3–99.2%) | 42 | 100% (91.6–100%) | 26 | 100% (87.1–100%) |
| | NPA | 217 | 94.0% (90.0–96.5%) | 222 | 89.6% (84.9–93.0%) | 201 | 99.5% (97.2–99.9%) | 217 | 93.1% (88.9–95.8%) |
| Homogeneous | PPA | 54 | 81.5% (69.2–89.6%) | 56 | 69.6% (56.7–80.1%) | 49 | 91.8% (80.8–96.8%) | 54 | 75.9% (63.1–85.4%) |
| | NPA | 189 | 100% (98.0–100%) | 187 | 98.9% (96.2–99.7%) | 194 | 98.5 (95.6–99.5%) | 189 | 99.5% (97.1–99.9%) |
| Speckled | PPA | 45 | 91.1% (79.3–96.5%) | 47 | 72.3% (58.2–83.1%) | 53 | 83.0% (70.8–90.8%) | 45 | 86.7% (73.8–93.7%) |

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| Positive and negative pattern agreement on HEp-2 Elite IFA substrate, compared to predicate for individual readers (Readers 1–3), and for consensus read of 2/3 readers. $n$ values depict samples positive or negative by the predicate device. | | | | | | | | | |
| --- | --- | --- | --- | --- | --- | --- | --- | --- | --- |
| Pattern | | HEp-2 Elite IFA v Predicate ANA IFA | | | | | | | |
| | | Reader 1 | | Reader 2 | | Reader 3 | | Consensus (≥2/3 Readers) | |
| | NPA | 198 | 99.5% (97.2–99.9%) | 196 | 98.0% (94.9–99.2%) | 190 | 96.3% (92.6–98.2%) | 198 | 99.5% (97.2–99.9%) |

*denominator values for “all” pattern reads are greater than 243, as these represent all pattern calls, irrespective of pattern multiplicity. e.g. samples with two patterns are counted twice

** see DFS(70) pattern agreement tables below, for more detailed analysis

To specifically represent the DFS70 pattern agreement data in more detail, the data in the Method Comparison table above are also depicted in the following $2 \times 2$ contingency tables, with PPA/NPA calculations, including $95\%$ CI:

| Reader 1 DFS(70) pattern agreement on HEp-2 Elite IFA substrate, compared to predicate for individual readers | | | | | |
| --- | --- | --- | --- | --- | --- |
| | Predicate | | | PPA: 100% (87.7–100%)
NPA: 94.0% (90.0–96.5%) | |
| | | DFS(+) | | | DFS(−) |
| HEp-2 Elite | DFS70(+) | 26 | 13 | | 39 |
| | DFS70(−) | 0 | 204 | | 204 |
| | | 26 | 217 | | 243 |
| Reader 2 DFS(70) Pattern Agreement | | | | | |
| | Predicate | | | PPA: 95.2% (77.3–99.2%)
NPA: 89.6% (84.9–93.0%) | |
| | | DFS(+) | | | DFS(−) |
| HEp-2 Elite | DFS70(+) | 20 | 23 | | 43 |
| | DFS70(−) | 1 | 199 | | 200 |
| | | 21 | 222 | | 243 |
| Reader 3 DFS(70) Pattern Agreement | | | | | |
| | Predicate | | | PPA: 100% (91.6–100%)
NPA: 99.5% (97.2–99.9%) | |
| | | DFS(+) | | | DFS(−) |
| HEp-2 Elite | DFS70(+) | 42 | 1 | | 43 |
| | DFS70(−) | 0 | 200 | | 200 |
| | | 42 | 201 | | 243 |

From these calculations, several samples were called DFS(−) on the predicate, but DFS70(+) on the HEp-2 Elite IFA substrate by Readers 1 and 2. Among these samples that were called DFS(−) on the predicate, but DFS70(+) on the HEp-2 Elite IFA substrate, approximately similar numbers were called as Homogeneous or Speckled on the predicate and negative for Homogeneous or Speckled on the Hep-2

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Elite IFA.: Reader 1 Homogeneous Predicate $(+)$ Elite $(-)=10$ ; Reader 2 Homogeneous Predicate $(+)$ Elite $(-)=17$ ; Reader 2 Speckled Predicate $(+)$ Elite $(-)=13$ .

In addition, pattern identity calls were compared between individual readers on the HEp-2 Elite IFA device and presented in the table below:

| Positive and negative pattern agreement on HEp-2 Elite IFA substrate, in pairwise comparisons of three readers. n values depict samples positive or negative by both readers. | | | | | | | |
| --- | --- | --- | --- | --- | --- | --- | --- |
| Pattern | | Reader 1 v Reader 2 | | Reader 1 v Reader 3 | | Reader 2 v Reader 3 | |
| | | n | % (95% CI) | n | % (95% CI) | n | % (95% CI) |
| All* | PPA | 181 | 92.1% (87.2–95.2%) | 179 | 73.8% (67.6–79.1%) | 173 | 72.4% (66.2–77.9%) |
| | NPA | 81 | 78.8% (69.0–86.2%) | 103 | 73.8% (61.6–83.2%) | 106 | 77.6% (65.3–86.4%) |
| DFS70 | PPA | 43 | 88.4% (75.5–94.9%) | 43 | 86.1% (72.7–94.5%) | 43 | 88.4% (75.5–94.9%) |
| | NPA | 200 | 99.5% (97.2–99.9%) | 200 | 99.0% (96.4–99.7%) | 200 | 97.5% (94.3–98.9%) |
| Homogeneous | PPA | 41 | 97.6% (87.4–99.6%) | 48 | 83.3% (70.4–91.3%) | 48 | 83.3% (70.4–91.3%) |
| | NPA | 202 | 98.0% (95.0–99.2%) | 195 | 97.9% (94.8–99.2%) | 195 | 99.5% (97.2–99.9%) |
| Speckled | PPA | 38 | 100% (90.8–100%) | 51 | 70.6% (57.0–81.3%) | 51 | 66.7% (53.0–78.0%) |
| | NPA | 205 | 98.0% (95.1–99.2%) | 192 | 96.9% (93.4–98.6%) | 192 | 97.9% (94.8–99.2%) |

*denominator values for "all" pattern reads are greater than 243, as these represent all pattern calls, irrespective of pattern multiplicity. e.g. samples with two patterns are counted twice

# b. Matrix comparison:

Not applicable; serum is the only sample matrix.

# 3. Clinical studies:

# a. Clinical Sensitivity and Specificity:

Tests for anti-nuclear antibodies aid in the diagnosis of SLE among other systemic autoimmune rheumatic diseases (SARD). A total of 256 diagnosed SARD samples were tested on the HEp-2 ELITE IFA. Non-ANA-associated disease conditions, including non-SARD autoimmune diseases, inflammatory diseases, cancer, and infectious diseases may be included in the intended use differential diagnosis for ANA. For comparison to the SARD sample cohort above, 254 samples of non-SARD autoimmune disease controls, 13 inflammatory disease controls, 57 cancer, and 173 samples from infectious disease cases were studied on the HEp-2 ELITE IFA.

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Sensitivity and specificity were calculated from these 753 total samples as below:

| SARD diseases and controls tested by the HEp-2 Elite | | | | |
| --- | --- | --- | --- | --- |
| | SARD | | | |
| | | (+) | | (-) |
| HEp-2 Elite | ANA(+) | 200 | 124 | 324 |
| | ANA(-) | 56 | 373 | 429 |
| | | 256 | 497 | 753 |

Sensitivity (95% CI): 78.1% (72.7 - 82.8%)

Specificity (95% CI): 75.1% (71.1 - 78.7%)

Performance of the HEp-2 Elite IFA for individual diseases are given for individual diagnostic categories below:

| Reported ANA-positive n, and percent positive on HEp-2 Elite IFA with disease samples | | | |
| --- | --- | --- | --- |
| Disease/Indication | Total n | Positive | |
| | | n | % |
| ANA-associated SARD samples | 256 | 200 | 78.1 |
| Autoimmune hepatitis | 13 | 6 | 46.2% |
| Antiphospholipid syndrome | 15 | 7 | 46.7% |
| Drug-induced lupus | 12 | 11 | 91.7% |
| Mixed connective tissue disease | 10 | 7 | 70% |
| Myositis | 36 | 16 | 44.4% |
| Sjögren's syndrome* | 65 | 62 | 95.3% |
| Systemic lupus erythematosus* | 78 | 74 | 94.9% |
| Systemic sclerosis* | 27 | 17 | 63.0% |
| Autoimmune disease controls | 254 | 90 | 35.4% |
| ANCA-associated vasculitis | 20 | 1 | 5% |
| Celiac disease | 10 | 1 | 10% |
| Crohn's disease | 25 | 6 | 24% |
| Autoimmune thyroiditis | 20 | 1 | 5% |
| Pernicious anemia | 20 | 2 | 10% |
| Primary biliary cholangitis | 25 | 17 | 68% |
| Psoriasis | 20 | 2 | 10% |
| Raynaud's syndrome, primary | 4 | 2 | 50% |
| Rheumatoid arthritis* | 87 | 52 | 59.8% |
| Ulcerative colitis | 23 | 6 | 26.1% |
| Inflammatory disease controls | 13 | 2 | 15.4% |
| Atopic dermatitis | 13 | 2 | 15.4% |
| Cancer | 57 | 3 | 5.3% |
| Breast cancer | 8 | 1 | 12.5% |
| Colorectal cancer | 10 | 1 | 10% |
| Ovarian cancer | 10 | 0 | 0% |
| Pancreatic cancer | 9 | 0 | 0% |
| Prostate cancer | 20 | 1 | 5% |
| Infectious disease controls | 173 | 29 | 16.8% |

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| Reported ANA-positive n, and percent positive on HEp-2 Elite IFA with disease samples | | | |
| --- | --- | --- | --- |
| Disease/Indication | Total n | Positive | |
| | | n | % |
| Cytomegalovirus | 20 | 1 | 5% |
| Hepatitis B virus | 20 | 1 | 5% |
| Hepatitis C virus* | 11 | 4 | 36.4% |
| HIV | 20 | 2 | 10% |
| Herpes simplex virus | 20 | 2 | 10% |
| Lyme disease | 12 | 6 | 50% |
| Mononucleosis | 10 | 2 | 20% |
| Rubella | 20 | 4 | 20% |
| Syphilis | 20 | 2 | 10% |
| Toxoplasmosis | 20 | 5 | 25% |
| Total: | 753 | 324 | 43.0% |

*cohort includes secondary Raynaud's syndrome sample(s), subordinate to primary diagnosis

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

Not applicable

4. Clinical cut-off

See Assay cut-off, above

5. Expected values/Reference range:

A cohort of 128 normal human serum samples was tested on the HEp-2 Elite IFA at the 1:40 initial dilution. Qualitative results demonstrating ANA prevalence in reference populations for this study are below:

| Reported ANA-positive n, and percent positive on HEp-2 Elite IFA on normal human samples | | | |
| --- | --- | --- | --- |
| Patient Status | Total n | Positive | |
| | | n | % |
| normal human sera | 128 | 7 | 5.5% |

Of the seven ANA-positive samples, three were DFS(+), two were AMA(+), and two were centromere(+).

N. Proposed Labeling:

The labeling is sufficient and it satisfies the requirements of 21 CFR Parts 801 and 809, as applicable.

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

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