This test system is for in vitro diagnostic use for the detection of antinuclear antibodies in human serum, with specific identification of autoantibodies to the SS-A/Ro antigen. This test system is to be used as an aid in the detection of antibodies associated with systemic rheumatic disease. The results from this assay can be used as an aid in the diagnosis of autoimune diseases.

Device Description

This is an indirect fluorescent antibody test for the semi-quantitative detection of antinuclear antibody in human serum. The transfected HEP-2 cell line allows for the identification of anti-SS-A/Ro antibodies because of the unique staining pattern that these antibodies show on this cell line.

AI/ML Overview

Here's an analysis of the provided text, outlining the acceptance criteria and study details for the HEp-2000® Fluorescent ANA-Ro Test System:

Acceptance Criteria and Device Performance Study

The provided text describes a submission for a new device, the HEp-2000® Fluorescent ANA-Ro Test System, seeking substantial equivalence to a legally marketed device (RELISA® SS-A/SS-B Antibody Test System, K955603). The study focuses on demonstrating the unique ability of the HEp-2000® cell line to specifically identify anti-SS-A/Ro antibodies and confirming the presence of SS-A/Ro antibodies.

1. Table of Acceptance Criteria and Reported Device Performance

The acceptance criteria are implied through the comparisons to existing HEp-2 cells and ELISA testing, specifically highlighting the ability to identify SS-A/Ro antibodies distinctly.

Acceptance Criteria (Implied)	Reported Device Performance (HEp-2000® Fluorescent ANA-Ro Test System)
Normal Samples:	Normal Samples (500 healthy blood donors):
- Low false positive rate for ANA	- 36 samples (7.2%) showed positive ANA reactions at 1:40 dilution.
- Consistent patterns with non-transfected HEp-2 where applicable.	- Patterns identical on both substrates for 34 of 36 positives.
- Identify SS-A/Ro in true SS-A/Ro positive samples even if non-transfected HEp-2 is negative/weak.	- Two discrepant samples (both SS-A/Ro confirmed by ELISA and Western blot): One showed weak fine speckled on non-transfected HEp-2 but typical "SS-A/Ro" staining on HEp-2000®. The other was negative on non-transfected HEp-2 but showed typical "SS-A/Ro" staining on HEp-2000®.
Sera with only Ro/SS-A Antibodies:	Sera from 46 patients with only Ro/SS-A antibodies:
- High sensitivity for SS-A/Ro antibodies	- All 46 (100%) were positive (distinctive Ro/SS-A staining pattern) with HEp-2000®.
- Superior performance to non-transfected HEp-2 for SS-A/Ro detection.	- 36 (78%) were positive (speckled pattern) with non-transfected HEp-2 cells.
Sera with other Autoantibodies:	Sera from 230 patients with various rheumatic/non-rheumatic diseases:
- Consistent patterns for non-SS-A/Ro antibodies.	- 333 staining patterns were identical on both substrates (single or mixed patterns).
- Accurate identification of SS-A/Ro antibodies even in complex samples.	- 29 samples showed distinctive "SS-A/Ro" staining pattern on HEp-2000®. 23 had speckled patterns on non-transfected HEp-2. The 6 discrepant samples (positive on HEp-2000®, negative on non-transfected HEp-2) all had SS-A/Ro antibodies confirmed by distinctive pattern, ELISA, and Western blot.
Titer Comparisons:	Titer Comparisons:
- Higher titers for SS-A/Ro on HEp-2000® (due to overexpression).	- Samples with anti-Ro/SS-A antibodies show higher titer values on HEp-2000® than on non-transfected HEp-2.
- No significant titer differences for other autoantibodies.	- Sera with other autoantibody specificities do not show significant titer differences.
Titer Reproducibility:	Titer Reproducibility (10 samples, 3 lots, 3 occasions):
- Consistent results across lots and runs.	- No negative sample showed positive results. All titer values within one two-fold dilution of established mean.
Confirmation of SS-A/Ro Antibodies:	Confirmation of SS-A/Ro Antibodies (349 known ANA positive samples):
- High positive predictive value for distinctive SS-A/Ro pattern.	- Of 239 samples with distinctive SS-A/Ro staining pattern, 238 (99.6%) had positive ELISA tests for SS-A/Ro antibodies.
- Absence of distinctive pattern does not rule out SS-A/Ro.	- An additional 79 samples with other patterns (speckled/homogeneous) gave positive ELISA tests for SS-A/Ro antibodies.

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

The "test set" in this context refers to the samples used to demonstrate the device's performance against the described criteria, likely corresponding to a validation or clinical study set.

Sample Sizes:
- Normal Samples: 500 healthy blood donors (242 females, 258 males).
- Sera from Patients with Only Ro/SS-A Antibodies: 46 patients with SLE or Sjögren's Syndrome.
- Sera from Patients with Autoantibodies Other Than SS-A/Ro: 230 patients with a variety of rheumatic and non-rheumatic diseases.
- Titer Reproducibility: 10 samples (CDC controls and in-house sera).
- Confirmation of SS-A/Ro Antibodies: 349 patients with known positive ANA tests (from a large rheumatology reference laboratory).
- Total SS-A/Ro containing sera examined across studies: 429 sera.
Data Provenance: The document does not explicitly state the country of origin. The mention of "CDC controls" (Centers for Disease Control and Prevention) suggests a US origin or at least use of US-standardized materials. Given the FDA submission, it's highly likely the studies were conducted in or accepted by the US regulatory framework. The data is retrospective, as it involves analyzing existing sera.

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

The document does not explicitly state the number or qualifications of experts used for establishing ground truth, such as interpreting the HEp-2 or HEp-2000 staining patterns. It implies that these interpretations were done by qualified laboratory personnel following established protocols for fluorescent antibody tests. However, the ground truth for the presence of SS-A/Ro antibodies was established through objective methods.

4. Adjudication Method for the Test Set

The document does not explicitly describe a formal adjudication method (like 2+1 or 3+1 consensus) for the interpretation of fluorescent patterns. The interpretation appears to be based on standard laboratory practice, with discrepancies or confirmations being resolved by "ELISA testing and by Western immunoblotting." This suggests a hierarchical approach rather than a consensus among multiple human readers for pattern interpretation.

5. Multi-Reader Multi-Case (MRMC) Comparative Effectiveness Study

No, a MRMC comparative effectiveness study was not explicitly described. The study compares the performance of the new device (HEp-2000®) against an older method (non-transfected HEp-2 cells) and a predicate ELISA device, but it doesn't involve multiple human readers interpreting cases with and without AI assistance to measure reader improvement. The "device" in this context is primarily an improved assay, not an AI-powered interpretation tool for human readers.

6. Standalone Performance Study

Yes, a standalone performance study was implicitly done. The entire submission describes the performance of the "HEp-2000® Fluorescent ANA-Ro Test System" (the algorithm/device) on its own, independent of human interpretation improvements (as it's a diagnostic test providing a result rather than an interpretative aid). The results presented are the direct output of the test system, compared to ground truth methods.

7. Type of Ground Truth Used

The ground truth for the presence or absence of SS-A/Ro antibodies was established by objective, highly specific laboratory methods:

ELISA testing (Predicate Device): RELISA® SS-A/SS-B Antibody Test System (K955603)
Western immunoblotting

For the patterns observed on HEp-2 and HEp-2000 cells, the ground truth is established by the accepted morphological characteristics of these patterns, which implicitly relies on expert consensus in the field for what constitutes a "speckled pattern" or "typical SS-A/Ro staining." However, when a discrepancy arose or confirmation was needed, the more objective ELISA and Western blot results trumped the pattern interpretation for confirming SS-A/Ro antibody presence.

8. Sample Size for the Training Set

The document does not specify a separate "training set" or its sample size. The entire dataset described appears to be used for validation and demonstration of performance. This is typical for a 510(k) submission for an in-vitro diagnostic assay improvement, where the "training" (development) of the cell line happened prior to these validation studies.

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

Since a separate "training set" is not detailed, the method for establishing ground truth for any underlying development of the HEp-2000® cell line is not described in this document. It can be inferred that the development of the transfected cell line was guided by scientific understanding of SS-A/Ro autoantigen expression and its visual manifestation in IF (Immunofluorescence) assays, likely confirmed by similar biochemical and immune-assay methods as used in the validation steps (e.g., ELISA, Western Blot, molecular characterization of the transfection).

Summary

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== Immuno
concepts

FEB 2 4 1998

STORY BOOK FAIR

Date Prepared: June 2, 1997 Contact Person: Eric S. Hoy, Ph.D. Name of Device:

· Trade Name HEp-2000® Fluorescent ANA-Ro Test System
· Common Name HEp-2000® Fluorescent ANA-Ro Test System
· Classification Name Antinuclear Antibody (21 CFR 866.5100)

Legally marketed device with which this device has been shown to be equivalent: RELISA® SS-A/SS-B Antibody Test System, K955603

Description:

Intended Use:

Summary of Technological Characteristics Compared to the Predicate Device: This device is a fluorescent antibody test for the detection of antinuclear antibodies. This 510(k) submission is an extension of K944096. In this new submission we have shown that the transfected HEp-2000® cell line produces a distinctive pattern that is confirmatory for the presence of antibodies to the SS-A/Ro autoantigen. We have compared the present device to an ELISA assay which is confirmatory for antibodies to the SS-A/Ro autoantigen (K955603).

Description of Laboratory Data That Indicate Substantial Equivalence: HORMAL SAMPLES

Sera from 500 healthy blood donors, 242 females and 258 males, none of whom had any known history of rheumatic diseases, were tested in parallel using commercially available, non-transfected HEp-2 cells and the EEp-2000@ Fluorescent ANA-Ro Test System. In this population, 36 samples (7.2%) showed positive antinuclear antibody tests at a 1:40 dilution of serum. Patterns of staining were identical on the two substrates for 34 of the 36 positive The two samples that showed differences were both from female samples. patients, and both were confirmed to contain anti-SS-A/Ro antibodies. One of

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these samples showed a weak fine speckled reaction on the non-transfected HBp-2 cells and the typical "SS-A/Ro" staining on the HEP-2000® Pluorescent ANA-Ro Test System. The other sample was negative on the non-transfected HBp-2 cells, but showed typical "SS-A/Ro" staining on the HEp-2000® Fluorescent ANA-Ro Test System. The SS-A/Ro specificity of these two samples was confirmed by BLISA testing and by Western immunoblotting.

SERA FROM PATIENTS WITH ONLY Ro/SS-A ANTIBODIES

Sera from 46 patients with SLB or Sjögren's Syndrome were tested using commercially available, non-transfected HEp-2 cells and the HEp-2000@ Fluorescent ANA-Ro Test System. All of these sera were confirmed to contain antibodies to the SS-A/Ro autoantigen by ELISA testing and by Western immunoblotting. No other autoantibodies were detected in any of these Thirty six of these samples (78%) were positive (speckled pattern) samples. with the non-transfected HEp-2 cells, and all 46 (100%) were positive (distinctive Ro/SS-A staining pattern) with the HEP-20000 Fluorescent ANA-Ro Test System.

SERA FROM PATIENTS WITH AUTOANTIBODIES OTHER THAN 88-A/RO Serum samples from 230 patients with a variety of rheumatic and non-rheumatic diseases were tested in parallel using commercially available, non-transfected HEp-2 cells and the HEp-2000@ Fluorescent ANA-Ro Test System. A single staining pattern was seen with 120 semples, and mixed patterns were seen with 110 samples. Among the total population of 230 samples, 333 staining patterns were identical on both substrates. Twenty nine samples showed the distinctive "SS-A/Ro" staining pattern on the HBp-2000@ Test System. Twenty three of these samples showed speckled patterns on the non-transfected HEp-2 cells. The six discrepant samples (positive on HEp-20000, but negative on nontransfected HEp-2 cells) all had SS-A/Ro antibodies, as demonstrated by the distinctive "SS-A/Ro" staining pattern, ELISA tests, and Western blot confirmation.

TITER COMPARISONS

Because of the overexpression of the SS-A/Ro autoantigen in the HEp-2000® cells, samples that contain anti-Ro/SS-A antibodies show higher titer values on these cells than the values obtained on non-transfected HBp-2 cells. Since none of the other autoantigens in the HBp-2000® cells are affected by the transfection process, sera with other autoantibody specificities do not show significant titer differences between the transfected HER-2000® cell line and non-transfected HEp-2 cells.

TITER REPRODUCIBILITY

Ten samples, chosen from CDC controls and other well characterized in-house sera, were run on three different lot numbers of HED-2000® slides, on three different occasions. In no case did a negative sample show positive results. All titer values were within one two-fold dilution of the established mean titer value for all samples tested.

CONFIRMATION OF 88-A/RO ANTIBODIES

In a large rheumatology reference laboratory, serum samples from 349 patients with known positive ANA tests were assayed using the HBp-2000® Fluorescent Teat System. In this selected population, 239 samples showed the distinctive SS-A/Ro staining pattern. Positive ELISA tests for SS-A/Ro antibodies were obtained in 238 (99.68) of these samples. An additional 79 samples showed

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strong speckled and/or homogeneous patterns, but gave positive ELISA tests for SS-A/Ro antibodies.. Thus, if the distinctive SS-A/Ro pattern is seen, it is confirmatory for the presence of SS-A/Ro antibodies, but the absence of the pattern does not rule out the possible presence of SS-A/Ro antibodies.

In the studies outlined above, we have examined a total of 429 sera that contain SS-A/Ro antibodies as confirmed by ELISA testing and/or Western Immunoblots, and which showed the distinctive SS-A/Ro staining pattern on the transfected HEp-2000® cell line. We have also seen samples which contain SS-A/Ro antibodies, but do not display the distinctive SS-A/Ro staining pattern, because high levels of other autoantibodies (usually anti-DNA antibodies or anti-Sm/RNP antibodies) mask the SS-A/Ro pattern. Thus, if the distinctive SS-A/Ro pattern is seen, it is confirmatory for the presence of SS-A/Ro antibodies, but the absence of the pattern does not rule out the possible presence of SS-A/Ro antibodies.
In accordance with 21 CFR 807.92(b)(3), we conclude from these data that the present device is substantially equivalent to the predicate device.

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Image /page/3/Picture/1 description: The image shows the seal of the Department of Health & Human Services USA. The seal features a stylized eagle with three lines forming its body and wings. The words "DEPARTMENT OF HEALTH & HUMAN SERVICES USA" are arranged in a circular pattern around the eagle.

Food and Drug Administration 2098 Gaither Road Rockville MD 20850

Eric S. Hoy, Ph.D. Chief, Scientific Officer Immuno Concepts, Inc. 9779 "D" Business Park Drive Sacramento, California 95827

FEB 2 4 1998

K972145/S2 Re : Hep-2000® Fluorescent ANA-Ro Test System Trade Name: Requlatory Class: II Product Code: DHN February 4, 1998 Dated: Received: February 6, 1998

Dear Dr. Hoy:

We have reviewed your Section 510(k) notification of intent to market the device referenced above and we have determined the device is substantially equivalent (for the indications for use stated in the enclosure) to 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, Druq, and Cosmetic Act (Act). 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 reqistration, listing of devices, good manufacturing practice, labeling, and prohibitions against misbranding and adulteration.

If your device is classified (see above) into either class II (Special Controls) or class III (Premarket Approval), it may be subject to such additional controls. Existing major regulations affecting your device can be found in the Code of Federal Regulations, Title 21, Parts 800 to 895. A substantially equivalent determination assumes compliance with the current Good Manufacturing Practice requirement, as set forth in the Quality System Requlation (QS) for Medical Devices: General regulation (21 CFR Part 820) and that, through periodic (QS) inspections, the Food and Druq Administration (FDA) will verify such assumptions. Failure to comply with the GMP regulation may result in regulatory action. In addition, FDA may publish further announcements concerning your device in the Federal Reqister. Please note: this response to your premarket notification submission does not affect any obligation you might have under sections 531 through 542 of the Act for devices under the Electronic Product Radiation Control provisions, or other Federal Laws or Requlations.

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Page 2

Under the Clinical Laboratory Improvement Amendments of 1988 (CLIA-88), this device may require a CLIA complexity categorization. To determine if it does, you should contact the Centers for Disease Control and Prevention (CDC) at (770)488-7655.

This letter will allow you to begin marketing your device as described in your 510(k) premarket notification. The FDA finding of substantial equivalence of your device to a legally marketed predicate device results in a classification for your device and thus, permits your device to proceed to the market.

If you desire specific advice for your device on our labeling regulation (21 CFR Part 801 and additionally 809.10 for in vitro diagnostic devices), please contact the Office of Compliance at (301) 594-4588. Additionally, for questions on the promotion and advertising of your device, please contact the Office of Compliance at (301) 594-4639. Also, please note the regulation entitled, "Misbranding by reference to premarket notification" (21 CFR 807.97). Other general information on your responsibilities under the Act may be obtained from the Division of Small Manufacturers Assistance at its toll free number (800) 638-2041 or at (301) 443-6597 or at its internet address "http://www.fda.gov/cdrh/dsmamain.html"

Sincerely yours,

Steven Putman

Steven I. Gutman, M.D., M.B.A. Director Division of Clinical Laboratory Devices Office of Device Evaluation Center for Devices and Radiological Health

Enclosure

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Page_

510(k) Number (if known):

Device Name: HEp-2000 Fluorescent ANA-Ro Test System

Indications For Use:

This is an indirect fluorescent antibody test for the semiquantitative detection of antinuclear antibodies in human serum, with specific identification of autoantibodies to the SS-A/Ro This test system is to be used as an aid in the detection antigen. of antibodies associated with system rheumatic disease.

(PLEASE DO NOT WRITE BELOW THIS LINE-CONTINUE ON ANOTHER PAGE IF NEEDED)

Concurrence of CDRH, Office of Device Evaluation (ODE) Prescription Use N Over-The-Counter Use _________________________________________________________________________________________________________________________________________________________ OH (Fer 21 CFR 801 . 109)

(Optional Format *1-2-96)

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).