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 Description

The ImmuGlo™ HEp-2 Elite IFA is an indirect immunofluorescence antibody test for the qualitative detection of anti-nuclear antibodies (ANA) of the IgG isotype in human serum utilizing standard HEp-2 cells as a substrate. The HEp-2 Elite sides provided with this kit contain a 1:9 mixture of standard HEp-2 cells and engineered HEp-2 cells with the PSP1 gene knocked out. The engineered HEp-2 cells are able to detect all ANA specificities with the exception of the DF570/AC-2 pattern resulting from autoantibodies associated with PSP2. In DF570 positive reactions standard HEp-2 cells provide a positive reaction while engineered HEp-2 cells do not. The HEp-2 Elite substrate thereby provides additionality to aid in discriminating homogeneous, speckled, and dense fine speckled (DFS70/AC-2) patterns during the screening phase.

AI/ML Overview

The provided text describes the ImmuGlo HEp-2 Elite IFA device, which is an indirect immunofluorescence antibody test for the detection of anti-nuclear antibodies (ANA). It aims to aid in the diagnosis of systemic rheumatic diseases.

Here's an analysis of the acceptance criteria and the study data:

1. Table of Acceptance Criteria and Reported Device Performance

The document does not explicitly state formal acceptance criteria in a dedicated section for all tests. However, performance metrics are provided in the "Non-clinical Tests" and "Precision" sections. Based on the "Repeatability" and "Reproducibility" sections, the acceptance criteria for precision studies are implicitly defined.

Performance Metric	Acceptance Criteria (Stated or Implied)	Reported Device Performance
Qualitative Agreement (Pos/Neg)	Not explicitly stated as acceptance criteria, but implied performance goal for substantial equivalence.	Positive % Agreement: 99.7% (95% CI 97.8% - 100.0%) Negative % Agreement: 98.3% (95% CI 95.8% - 99.4%) Overall % Agreement: 99.3% (95% CI 97.8% - 99.5%) (vs. Predicate device)
Pattern Agreement Study (Overall)	Not explicitly stated as acceptance criteria, but implied performance goal compared to the predicate device.	Consensus (2/3 Readers) PPA: 88.9% (83.1-92.9%) Consensus (2/3 Readers) NPA: 76.2% (63.5-85.6%)
Pattern Agreement Study (DFS70)	Not explicitly stated.	Consensus (2/3 Readers) PPA: 100.0% (84.0-100.0%) Consensus (2/3 Readers) NPA: 93.1% (88.6-95.9%)
Repeatability	90% qualitative agreement (positive), fluorescence intensity agreement (± 1 fluorescence value within one run), and pattern agreement.	All samples met the 90% acceptance criteria for qualitative agreement (positive), fluorescence intensity agreement (± 1 fluorescence value within one run) and pattern agreement. (Total 60 replicates per sample, two operators, 120 total reads for 8 negative and 17 positive samples)
Reproducibility	AQL: ± 1 fluorescence value, ± 1 titer, and a consensus pattern agreement. Acceptance criteria: 95% or greater agreement (titer and fluorescence within ± 1 value, with consensus pattern).	Overall agreement was 99.75%. Two samples (6 and 8 for Homogeneous pattern and Titer respectively) did not meet AQL due to subjective reader error, but other patterns showed 93-100% Pattern Agreement, 93-100% Titer Agreement, and 100% Fluorescence Intensity Agreement. (For 11 samples, tested in triplicate in 10 runs at 3 sites, total 90 replicates per sample).
ANA Associated Disease Sensitivity	Not explicitly stated as acceptance criteria, but implied performance goal for clinical utility.	78.1% (95% C.I. 72.5-82.9%)
ANA Associated Disease Specificity	Not explicitly stated as acceptance criteria, but implied performance goal for clinical utility.	75.1% (95% C.I. 71.0-78.7%)

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

Qualitative Agreement (Pos/Neg) Study (vs. Predicate):
- Sample Size: 591 samples (298 positive, 293 negative by predicate).
- Data Provenance: Not explicitly stated, but the "Method Comparison" implies these are clinical samples tested against the predicate device. No details on country of origin or retrospective/prospective collection are provided.
Pattern Agreement Study:
- Sample Size: 243 samples with known ANA patterns.
- Data Provenance: Not explicitly stated. The samples were "randomized." No details on country of origin or retrospective/prospective collection are provided.
Repeatability Study:
- Sample Size: 8 negative and 17 positive samples. Each sample was tested in 6 replicates in 10 runs (over 5 days), resulting in 60 replicates for each sample. Read by 2 operators, total N = 120 per sample series.
- Data Provenance: Not specified.
Reproducibility Study:
- Sample Size: 10 positive samples with various patterns and intensities. Each sample was tested in triplicate in 10 runs (2 runs/day for 5 days) at 3 different sites, resulting in 90 replicates per sample.
- Data Provenance: Not specified, other than "three different sites."
Clinical Study (Sensitivity/Specificity):
- Sample Size: 256 ANA-associated disease samples and 497 autoimmune and infectious disease controls. (Total 753 samples, excluding normal human sera and DFS70 suspected samples for these specific calculations).
- Data Provenance: Not explicitly stated regarding country of origin or retrospective/prospective collection. These are referred to as "sets of clinical samples."

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

Qualitative Agreement Study: Ground truth was established by comparison to a "Predicate" device. No human experts are explicitly mentioned for ground truth.
Pattern Agreement Study: Three independent evaluators provided reads. The ground truth for the comparison was established by "Consensus (2/3 Readers)." The qualifications of these evaluators are not specified (e.g., if they are expert medical professionals like radiologists or pathologists, or their years of experience).
Precision (Repeatability and Reproducibility) Studies: The repeatability study mentions "2 operators." The reproducibility study mentions testing at "three different sites" and implies readers/operators at those sites. However, the qualifications of these operators/readers are not specified.
Clinical Study (Sensitivity/Specificity): The classification of samples into "ANA-associated autoimmune disease" or "control" implies a clinical diagnosis was used as ground truth, likely established by referring clinicians. However, the exact method of ground truth establishment (e.g., based on clinical criteria, pathology, or expert consensus) and the qualifications of those who established it are not detailed.

4. Adjudication Method for the Test Set

Qualitative Agreement (Pos/Neg) Study: The comparison is made against a "Predicate" device. No explicit human adjudication method for the ground truth of the samples themselves is stated; the predicate device's results are used as the reference.
Pattern Agreement Study: The adjudication method for comparing the Elite device against the predicate was based on "Consensus (2/3 Readers)." This means that for a result to be considered consensus, at least two out of the three independent evaluators had to agree.
Precision (Repeatability and Reproducibility) Studies: For reproducibility, "consensus pattern agreement" was part of the AQL. This indicates an adjudication process was used, likely involving agreement among multiple readers/operators, but the specific number or method isn't detailed beyond the term "consensus."

5. If a Multi-Reader Multi-Case (MRMC) Comparative Effectiveness Study Was Done

Yes, a form of MRMC study was done for pattern agreement. The "Pattern Agreement Study" involved 243 samples and 3 independent evaluators (readers) who were blinded to each other's results and the samples. The data was analyzed by comparing the Elite device vs. the Predicate device based on reader consensus (at least 2 of 3 readers).
Effect Size of Human Readers Improvement with AI vs. without AI assistance: This information is not applicable as the device (ImmuGlo HEp-2 Elite IFA) is a diagnostic kit read by human operators (microscopists/technologists) and is not described as an AI-powered image analysis tool. The study assesses the agreement between patterns identified using the new device and the predicate by human readers, not the effect of AI assistance on human reader performance.

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

No, the device described is an "indirect immunofluorescence antibody test kit" (IFA). This is a laboratory diagnostic method that requires human-in-the-loop performance for slide preparation, reading under a fluorescence microscope, and interpretation of patterns and titers. There is no mention of an algorithm-only or AI-driven standalone component in the provided text.

7. The Type of Ground Truth Used

Qualitative Agreement Study: The ground truth for this comparison was the results obtained from the predicate device.
Pattern Agreement Study: The ground truth for comparison in the pattern study was established by expert consensus among 2 out of 3 independent evaluators.
Clinical Study (Sensitivity/Specificity): The ground truth was based on a clinical diagnosis of "ANA-associated autoimmune disease" or "control" conditions. This typically reflects real-world clinical outcomes and physician diagnoses, potentially supported by other laboratory and clinical data. There is no mention of pathology or other specific "gold standard" laboratory tests cited as the primary ground truth.

8. The Sample Size for the Training Set

The document describes performance studies for a diagnostic kit (ImmuGlo HEp-2 Elite IFA). This typically does not involve "training sets" in the context of machine learning or AI models, but rather clinical samples are used for method comparison, precision, and clinical performance evaluation.
Therefore, a "training set" for an algorithm is not applicable to this device as described. The samples mentioned in the clinical study (753 samples) are test sets for evaluating the performance of the device itself.

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

As a "training set" in the AI or machine learning sense is not applicable to this device, the method for establishing its ground truth is not relevant here.

Summary

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June 05, 2018

IMMCO Diagnostics, Inc. Kevin Lawson Chief Regulatory Officer 60 Pineview Drive Buffalo, New York 14228

Re: K172745

Trade/Device Name: ImmuGlo HEp-2 Elite IFA Regulation Number: 21 CFR 866.5100 Regulation Name: Antinuclear antibody immunological test system Regulatory Class: Class II Product Code: DHN Dated: September 8, 2017 Received: September 12, 2017

Dear Kevin Lawson:

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 Part 801 and Part 809); medical device reporting of medical device-related adverse events) (21 CFR 803); good manufacturing practice requirements as set forth in the quality systems (QS) regulation (21 CFR

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

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 comprehensive regulatory information about medical devices and radiation-emitting products, including information about labeling regulations, please see Device Advice (https://www.fda.gov/MedicalDevices/DeviceRegulationandGuidance/) and CDRH Learn (http://www.fda.gov/Training/CDRHLearn). Additionally, you may contact the Division of Industry and Consumer Education (DICE) to ask a question about a specific regulatory topic. See the DICE website (http://www.fda.gov/DICE) for more information or contact DICE by email (DICE@fda.hhs.gov) or phone (1-800-638-2041 or 301-796-7100).

Sincerely.

Leonthena R. Carrington -S

Lea Carrington 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) K172745

Device Name ImmuGlo HEp-2 Elite IFA

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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ImmuGlo HEp-2 Elite IFA 510(k) Summary

Submitter:	Immco Diagnostics, Inc.
Address:	60 Pineview Dr., Buffalo, NY 14228
Phone Number:	716-691-0091 ext. 110
Contact:	Kevin Lawson
Summary Prepared:	6-5-2018
Device Name:	ImmuGlo™ HEp-2 Elite IFA
Common Name:	HEp-2 IFA
Product Code:	Antinuclear Antibody, Indirect Immunofluorescent, Antigen, Control [DHN
Substantially Equivalent to:	ImmuGlo ANTINUCLEAR ANTIBODY (ANA) TEST (HEP-2 CELLS)

General Description: Antinucles (ANA) target various nuclear and cellular components and are highly prevalent in systemic autoimmune connective tissue disorders. ANA can be detect immunofluorescence assay (IFA) using HEp-2 cell substrates. Such assys aid in the diagnosis of systemic lupus erythematosus (SLE) and other including mixed connective tissue disease, Sjögren's syndrome, systemic sclerosis, polymyositis, drug induced lupus, as well as cases of autoimmune hepatitis. ANA occur in about 95% of SLE patients. HEp-2 cell lines present a variety of autoantigens that are not found in solid phase assays of ANA. Solid phase assays developed on various platforms differ in their and specificity raising uncertainties regarding the interpretation of results. Due to the limitations of solid phase assays, the American College of Rheumatology (ACR) considers IFA using a HEp-2 cell substrate as the gold standard for ANA testing.

The HEp-2-lFA method is able to report both ANA pattern and titer for the test specimen. Various groups have worked to improve awareness of ANA patterns detected by HEp-2, criteria for accurate interpretation of ANA patterns. Much of this work has been compiled in the International Consensus on Antibody Patterns (CAP) report recommending a standardized nomenclature and reporting format for IFA patterns.

The major nuclear patterns observed on HEp-2 substrates 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 and AC-10) patterns. A critical development in recent years is the definition of a frequently reported pattern termed "Dense Fine Speckled" that has been assigned the anti-cell pattern number AC-2 and recommended as a competent level reporting parameter by CAP. The major autoantigenic target associated with the DFS/AC-2 pattern, also referred to as the DF570 pattern, is a 70 kDa antigen known as LEDGF (Lens Epithelium Derived Growth Factor) or PSP1 gene product. These autoantibodies were initially reported to occur in sera from patients with positive ANA tests but no clinical evidence of systemic alsease (SARD) and do not have a distinct clinical association. The prevalence of DF570 autoantibodies, methods used to screen/confirm the DFS/AC-2 pattern (i.e. IFA or solidphase) and implications of inaccurate interpretation of DF570/DFS/AC-2 pattern have been presented in literature. The DFS/DFS70/AC-2 pattern may be difficult to read and can be mis-interpreted as homogeneous (associated with DNA, histones and nucleosomes), fine speckled, or a mix of homogeneous and fine speckled patterns, all of which are associated with systemic autoimmune diseases of the DFS70 pattern and the lack of commercially available assays that aid in the DFS/DF570/AC-2 pattern from mixed disease associated patterns increases the need for a method that may aid in distinction between ANA patterns at the screening stage without compromising the inherent advantages of the HEp-2 IFA method. The ICAP committee recommends all clinical labs report the DFS70 pattern.

HEp-2 Elite sides provided with this kit contain a 1:9 mixture of standard HEp-2 cells devoid of functional copies of PSP2 gene (UniGene Gene ID: 1116). The engineered HEp-2 cels are able to detect all ANA specificities with the exception of the DF570/AC-2 pattern resulting from autoantibodies associated with PSP2. In DF570 positive reactions standard HEp-2 cells provide a positive reaction while engineered HEp-2 cells do not. The HEp-2 Elite substrate thereby provides additionality to aid in discriminating homogeneous, speckled, and dense fine speckled (DFS70/AC-2) patterns during the screening phase.

Intended Use: The ImmuGlo™ HEp-2 Elite IFA is an indirect immunofluorescence antibody test for the qualitative detection of anti-nuclear antibodies (ANA) of the IgG isotype in human serum utilizing standard HEp-2 cells as a substrate. The ImmuGlo™ HEp-2 Elite FA is intended for use as an aid in the diseases in conjunction with other clinical and laboratory findings.

Similarities and Differences: Both kits use HEp-2 cells in and positive controls, serum diluent, wash solution, lgG conjugate and mounting medium to detect ANA IgG antibodies. Both kits use 1:40 screening dilutions and 30 minute incubation steps and slides are read on a fluorescence microscope. The ImmuGlo HEp-2 cells mixed with engineered HEp-2 with the PSP1 gene knocked out in 1:9 ratio while the ImmuGlo ANTINUCLEAR ANTIBODY (ANA) TEST utilizes only standard HEp-2 cells.

60 Pineview Drive USA Toll free (800) 537-8378 · tel. (716) 691-0091 · · fax (716) 691-0466

www.immco.com

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Non-clinical Tests:

Method Comparison: Both kits were tested with well-characterized ANA associated disease controls.

		Predicate
		Positive	Negative	Total
Hep-2	Positive	297	5	302
Elite	Negative	1	288	289
IFA	Total	298	293	591
Positive % Agreement 99.7% (95% CI 97.8% - 100.0%)
Negative % Agreement 98.3% (95% CI 95.8% - 99.4%)
Overall % Agreement 99.3% (95% CI 97.8% - 99.5%)

Qualitative Agreement (Pos/ Neg)

Pattern Agreement Study

An additional study was designed to detern agreement using 243 samples with known ANA patterns. These samples were randomized and sides provided to 3 independent evaluators. The readers were blinded to each others' results, as well as ever the samples. The data was analyzed by comparing Elite vs Predicate by read (at least 2 of 3 readers). In the case of mixed patterns where more than one pattern was observed, the dominant or primary pattern was analyzed.

		Elite vs Predicate
Pattern			Reader 1		Reader 2		Reader 3		Consensus(2/3 Readers)
		n	% (95% CI)	n	% (95% CI)	n	% (95% CI)	n	% (95% CI)
All	PPA	162	98.8%(95.1-99.8%)	144	95.8%(90.8-98.3%)	197	95.4%(91.2-97.8%)	180	88.9%(83.1-92.9%)
	NPA	81	81.5%(71.0-88.9%)	99	67.7%(57.4-76.5%)	46	78.2%(63.2-88.5%)	63	76.2%(63.5-85.6%)
DFS(70)	PPA	26	100.0%(84.0-100.0%)	21	95.2%(74.1-99.7%)	42	100.0%(89.6-100.0%)	26	100.0%(84.0-100.0%)
	NPA	217	94.4%(89.7-96.6%)	222	89.6%(84.7-93.2%)	201	99.5%(96.8-100.0%)	217	93.1%(88.6-95.9%)
Homogeneous	PPA	54	81.5%(68.1-90.3%)	57	68.4%(54.6-79.7%)	49	91.8%(79.5-97.4%)	55	76.4%(62.7-86.3%)
	NPA	189	100.0%(97.5-100.0%)	186	98.9%(95.7-99.8%)	194	98.5%(95.2-99.6%)	188	100.0%(97.5-100.0%)
Speckled	PPA	45	88.9%(75.2-95.8%)	47	72.3%(57.1-83.9%)	53	83.0%(69.7-91.5%)	40	97.5%(85.3-99.9%)
	NPA	198	99.9%(96.0-99.8%)	196	98.0%(94.5-99.3%)	190	96.3%(92.3-98.4%)	203	97.0%(93.4-98.8%)

NOTE: Above data depicts primary pattern only. Results will differ if secondary pattern is included. n values depict samples positive or negative by the predicate device.

OBSERVED PREVALENCE

Tests for nuclear antibodies are used to screen other immunologic disturbances. Sets of clinical samples were tested on the HEp-2 Elite IFA. Results demonstrating prevalence in the populations for this study are provided below.

Condition	n Tested	n Positive	% Positive
ANA-associated autoimmune disease	256	200	78.1%
Autoimmune hepatitis	13	6	46.2%
Antiphospholipid syndrome	15	7	46.7%
Mixed connective tissue disease	10	7	70.0%
Myositis	36	16	44.4%
Sjögren's	65	62	95.4%

60 Pineview Drive Toll free (800) 537-8378 fax (716) 691-0466

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Systemic lupus erythematosus	78	74	94.9%
Systemic sclerosis	27	17	63.0%
Drug-induced lupus	12	11	91.7%
Non-ANA-associated autoimmune disease	254	90	35.4%
Celiac	10	1	10.0%
Crohn's	25	6	24.0%
Pernicious anemia	20	1	5.0%
Primary biliary cirrhosis	25	17	68.0%
Rheumatoid arthritis	87	52	68.0%
Autoimmune thyroiditis	20	2	10.0%
Ulcerative colitis	23	6	26.1%
ANCA Vasculitis	20	1	5.0%
Raynaud's syndrome	4	2	50.0%
Psoriasis	20	2	10.0%
Inflammatory disease	13	2	15.4%
Atopic dermatitis	13	2	15.4%
Cancer	57	3	5.3%
Colorectal cancer	10	1	10.0%
Ovarian cancer	10	0	0.0%
Pancreatic cancer	9	0	0.0%
Breast cancer	8	1	12.5%
Prostate cancer	20	1	5.0%
Infectious Disease	173	29	16.8%
Cytomegalovirus	20	1	5.0%
Hepatitis C	11	4	36.4%
Herpes simplex virus	20	2	10.0%
Lyme	12	6	50.0%
Mononucleosis	10	2	20.0%
Rubella	20	4	20.0%
Syphilis	20	2	10.0%
Toxoplasmosis	20	5	25.0%
HIV	20	2	10.0%
HBV	20	1	5.0%
Normal human serum	128	7	5.5%

Cross Reactivity See Observed Prevalence section above.

Precision

Repeatability

To assess repeatability of the ImmuGlo™ HEp-2 Elite IFA and a manual microscope 8 negative and 17 positive samples with various patterns and intensities were tested in 6 replicates in 10 runs per day for 5 days), resulting in 60 replicates for each sample read by 2 operators for a total n of 120. All samples met the 90% acceptance criteria for qualitative agreement (positive), fluorescence intensity agreement (± 1 fluorescence value within one run) and pattern agreement.

Reproducibility

In another study, the ImmuGl" HEp-2 Elte IFA and a manual microscope 10 positive sample with various patterns and intensities tested in triplicate in 10 runs (2 runs per day for 5 days) at three different sites, resulting in 90 replicates for each sample. % pattern agreement, % titer agreement and % fluorescenent results are provided below. The AQL for these studies was ± 1 fluorescence value, ± 1 titer and a consensus pattern agreement. The acceptance criteria for these studies was 95% or greater and rs within ± 1 fluorescence value, ± 1 titer with consent. The two samples that did not meet AQL were the result of subjective reader error. Overall agreement was 99.75%. Samples 2 and 9 demonstrated secondary pattern and intensity for these samples. No report of a secondary pattern is considered agreement for all other samples.

60 Pineview Drive USA Toll free (800) 537-8378 ● tel. (716) 691-0091 ● fax (716) 691-0466

www.immco.com

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Sample	Pattern	EndpointTiter	FluorescenceIntensity	PatternAgreement	TiterAgreement	FluorescenceIntensityAgreement
1	DFS70	80	2+	100%	100%	100%
2*	Cytoplasmic	80	2+	100%	100%	100%
	Nuclear dots	80	2+	100%	100%	100%
3	Speckled	80	2+	100%	100%	100%
4	Centromere	80	2+	100%	100%	100%
5	Speckled	80	2+	100%	100%	100%
6	Homogeneous	40	1+	93%	100%	100%
7	DFS70	80	2+	100%	100%	100%
8	Homogeneous	160	3+	100%	93%	100%
9*	Cytoplasmic	1280	3+	100%	100%	100%
	Nuclear dots	1280	4+	100%	100%	100%
10	Nucleolar	1280	4+	100%	100%	100%
11	Negative	0	0	100%	100%	100%

Samples 2 and 9 are mixed pattern specificity, primary pattern is listed first.

Interference

Interference was studied by mixing sera with potentially interfering serum samples and studying deviation from expected results. No significant interference was demonstrated for the following substances at the levels indicated: hemoglobin (2 g/L), unconjugated bilirubin (342 µmo/(), rheumatoid factor (13 mmo/4), triglycerides (3.7 mmo/4), naproxen (2170 µmo/4), ibuprofen (2425 umol/L), prednisone (0.84 µmo/L), mycophernyate mofetil (14.01 µg/ml), hydroxychloroquine (388 ng/ml), cyclophosphamide (1437 µmol/L), rituximab (1.143 mg/ml) and Belimumab (0.939 mg/ml).

Clinical Study: Sets of clinical samples were tested on the Included 256 AM associated disease samples and 497 autoimmune and infectious disease controls.

ANA Associated Disease Sensitivity: 78.1% (95% C.I. 72.5-82.9%) ANA Associated Disease Specificity: 75.1% (95% C.I. 71.0-78.7%).

ANA associated diseases used for calculation of sensitis, Antiphospholipid syndrome, Mixed connective tissue disorder, Myositis, Siögren's syndrome, Systemic sclerosis and Drug induced lupus erythematosus. Disease Control samples (non-ANA associated diseases) used for calculation of specificity included Celiac disease, Pernicious anemia, Primary biliary cirrhosis, Rheumatoid arthritis, Autoimmune thyroid disease, Ulcerative colitis, Raynaud's syndrome, Psoriasis, Atopic dermatitis, Colorectal cancer, Ovarian cancer, Breast cancer, Prostate cancer, Cytomegalovirus, Hepatitis C, Herpes simplex virus, Lyme disease, Mononucleosis, Rubella, Syphilis, Toxoplasmosis, Human immunodeficiency virus and Hepattis B. Normal human sera and DF570 suspected samples were excluded in sensitivity/specificity calculations.

Stability: All reagents for this kit are the sredicate device except for the slide, so accelerated stability testing was performed on the slide to ensure no change in shelf life. Accelerated stability testing on 3 lots stored at 37℃ for up to 30 days showed no drop in fluorescence over the course of the experiment supporting the predicate shelf life of 18 months. Real time and open kit stability is currently 18 months for 2 lots and 9 for a third, which all continue to meet AQL.

Ken Burns

Kevin J. Lawson Chief Regulatory Officer

60 Pineview Drive Toll free (800) 537-8378 · tel. (716) 691-0091 · · fax (716) 691-0466

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Regulation Number and Section

§ 866.5100 Antinuclear antibody immunological test system.

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