← Product Code [KTL](/submissions/IM/subpart-f%E2%80%94immunological-test-systems/KTL) · K172252

# EUROIMMUN IFA: Crithidia luciliae sensitive (anti-dsDNA) EUROPattern (K172252)

_Euroimmun Us, Inc. · KTL · Apr 20, 2018 · Immunology · SESE_

**Canonical URL:** https://fda.innolitics.com/submissions/IM/subpart-f%E2%80%94immunological-test-systems/KTL/K172252

## Device Facts

- **Applicant:** Euroimmun Us, Inc.
- **Product Code:** [KTL](/submissions/IM/subpart-f%E2%80%94immunological-test-systems/KTL.md)
- **Decision Date:** Apr 20, 2018
- **Decision:** SESE
- **Submission Type:** Traditional
- **Regulation:** 21 CFR 866.5100
- **Device Class:** Class 2
- **Review Panel:** Immunology

## Indications for Use

The EUROIMMUN IFA: Crithidia luciliae sensitive (anti-dsDNA) EUROPattern test kit is intended for the qualitative and semiquantitative determination of human antibodies of immunoglobulin class IgG against anti-double stranded DNA (dsDNA) in human serum with the EUROPattern Microscope and Software automated instrument. It is a sensitive method, used as an aid in the diagnosis of systemic lupus erythematosus (SLE), in conjunction with other laboratory and clinical findings. All suggested results obtained with the EUROPattern system must be confirmed by trained personnel.

## Device Story

Device is an indirect immunofluorescence (IFA) assay using Crithidia luciliae substrate to detect anti-dsDNA antibodies in human serum. Patient samples are incubated on BIOCHIP slides containing Crithidia luciliae smears; specific antibodies bind to the kinetoplast (containing dsDNA). After washing, fluorescein-labeled anti-human IgG conjugate is added. The EUROPattern microscope and software system automates imaging and evaluation of fluorescence intensity. The system provides qualitative and semi-quantitative (titer) results. Used in clinical laboratories by trained personnel. The software provides automated analysis, but all results require confirmation by a trained operator. The device aids in SLE diagnosis by identifying anti-dsDNA reactivity, which is highly specific for the disease.

## Clinical Evidence

Clinical study evaluated 364 characterized samples (98 SLE, 266 non-SLE) across three sites. Sensitivity for SLE ranged from 45.9% to 48.6% depending on the evaluation mode (A, B, or C). Specificity ranged from 90.5% to 94.7%. Method comparison against the predicate ELISA showed 89.0% overall agreement. Precision/reproducibility studies (repeatability and site-to-site) demonstrated high titer agreement (within ±1 titer) and positive/negative agreement across automated and manual modes.

## Technological Characteristics

IFA test kit using Crithidia luciliae substrate; automated fluorescence microscope; software for image acquisition and analysis. Connectivity: integrated system. Software: automated pattern recognition/quantification.

## Regulatory 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

- EUROIMMUN Anti-dsDNA-NcX ELISA ([K103536](/device/K103536.md))

## Submission Summary (Full Text)

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>
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# 510(k) SUBSTANTIAL EQUIVALENCE DETERMINATION DECISION MEMORANDUM

A. 510(k) Number:
K172252

B. Purpose for Submission:
New Device on a previously cleared instrument

C. Measurand:
Anti-double stranded DNA (ds DNA) antibodies

D. Type of Test:
Qualitative and semi-quantitative

E. Applicant:
EUROIMMUN US Inc

F. Proprietary and Established Names:
EUROIMMUN IFA: Crithidia luciliae sensitive (anti-dsDNA) EUROPattern

G. Regulatory Information:

1. Regulation section:
21 CFR 866.5100, Antinuclear antibody immunological test system
21 CFR 866.4750, Automated indirect immunofluorescence microscope and software-assisted system for clinical use
21 CFR Parts 862-892, Single (specified) analyte controls (assayed and unassayed)

2. Classification:
Class II

3. Product code:
KTL, anti-DNA indirect immunofluorescent solid phase
PIV, automated indirect immunofluorescence microscope and software-assisted system for clinical use

4. Panel:
82, Immunology

H. Intended Use:

1. Intended use(s):
The EUROIMMUN IFA: Crithidia luciliae sensitive (anti-dsDNA) EUROPattern test kit is intended for the qualitative and semi-quantitative determination of human antibodies of immunoglobulin class IgG against anti-double stranded DNA (dsDNA) in human serum with

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the EUROPattern Microscope and Software automated instrument. It is a sensitive method, used as an aid in the diagnosis of systemic lupus erythematosus (SLE), in conjunction with other laboratory and clinical findings. All suggested results obtained with the EUROPattern system must be confirmed by trained personnel.

2. Indication(s) for use:
Same as intended use

3. Special conditions for use statement(s):
1. For prescription use only
2. This device is only for use with reagents that are indicated for use with the device.
3. The device is for use by a trained operator in a clinical laboratory setting.
4. All software-aided results must be confirmed by the trained operator.
5. Special instrument requirements: for use only with EUROPattern Microscope and Software ((cleared in K141827))

I. Device Description:

Materials provided. The kit contains enough for 50 determinations:
- Slides, each containing 5 BIOCHIPs coated with a smear of Crithidia luciliae
- Conjugate, Fluorescein-labelled anti-human IgG (goat), ready for use with Evans Blue
- Positive control: autoantibodies against dsDNA, human, ready for use
- Negative control: autoantibody-negative, human, ready for use
- Sample buffer 2 (Crithidia luciliae sensitive IFA)
- Salt for PBS pH 7.2
- Tween 20
- Mounting medium, glycerol, ready for use
- Cover glasses (62 mm x 23 mm)
- Instruction booklet

Needed and not provided:
- Reagent trays for slides containing up to 10 fields (order #ZZ 9999-0110)
- Fluorescence microscope (EUROPattern microscope & software): Equipped with a 450-490 nm excitation filter; 510 nm color separator & 515-565 nm bandpass filter with LED Bluelight
- Distilled or de-ionized water for wash buffer production
- Pipettes with a range of 10 µl to 200 µl
- Cuvettes or wash/staining dishes for PBS wash step
- Lint free towelling

J. Substantial Equivalence Information:

1. Predicate device name(s):
EUROIMMUN Anti-dsDNA-NcX ELISA (IgG)

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2. Predicate 510(k) number(s): K083381

3. Comparison with predicate:

|  Similarities  |   |   |
| --- | --- | --- |
|  Item | Device EUROIMMUN IFA: Crithidia luciliae (anti-dsDNA) EUROPattern | Predicate EUROIMMUN Anti-dsDNA-NcX ELISA (IgG) (K083381)  |
|  Intended Use | For the qualitative and semi-quantitative determination of human antibodies of immunoglobulin class IgG against anti-double stranded DNA (dsDNA) in human serum with the EUROPattern Microscope and Software automated instrument. It is a sensitive method, used as an aid in the diagnosis of systemic lupus erythematosus (SLE), in conjunction with other laboratory and clinical findings. All suggested results obtained with the EUROPattern system must be confirmed by trained personnel. | For the quantitative or qualitative determination of IgG class autoantibodies against double-stranded genomic DNA (dsDNA) in human serum and EDTA or citrate plasma. It is used as an aid in the diagnosis of systemic lupus erythematosus, in conjunction with other laboratory and clinical findings.  |
|  Controls | 1 Positive control 1 Negative control | Same  |
|  Procedure | Standard immunoassay: Serum incubation with antigen, followed by a wash step, incubation with labelled anti-human globulin, wash step, measurement/evaluation | Same  |
|  Differences  |   |   |
| --- | --- | --- |
|  Item | Device | Predicate  |
|  Technology | IFA | ELISA  |
|  Assay platform | BIOCHIP TITERPLANE technology | 96-well microtiter plates  |
|  Assay format | Qualitative and semi- | Qualitative or quantitative  |
|   | quantitative | quantitative  |

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|  Differences  |   |   |
| --- | --- | --- |
|  Item | Device | Predicate  |
|   | quantitative |   |
|  Antigen | Crithidia luciliae cells | dsDNA antigen complexed with nucleosomes (NcX)  |
|  Conjugate | Fluorescein labeled goat anti-human IgG | Horseraddish peroxidase-labeled rabbit anti-human IgG  |
|  Calibrators | None | Three calibrators, calibrated against WHO/NIBSC Wo/80 standard  |
|  Matrix | Serum | Serum, EDTA or citrate plasma  |
|  Sample dilution | 1:10 | 1:201  |
|  Measurement/ evaluation | Visual evaluation of fluorescence under microscope, IFA EUROPattern automatic evaluation with user verification | Photometric reading  |
|  Cutoff | Negative at <1:10 dilution Positive at 1:10 dilution | Sample OD / Cut-off calibrator OD or 3-point calibrated analysis Cut-off: Ratio 1.0 or 100 IU/ml  |
|  Reported results | Qualitative, Titer | Qualitative, IU/ml  |

## K. Standard/Guidance Document Referenced:
N/A

## L. Test Principle:
For the detection of autoantibodies against native, double-stranded DNA (dsDNA, nDNA) by indirect immunofluorescence, EUROIMMUN uses as the assay substrate the Crithidia luciliae species. This protozoon possesses a giant mitochondrium containing double-stranded DNA (kinetoplast) which shows none of the remaining antigens contained in human cell nuclei. Antinuclear antibodies reacting with the kinetoplast are directed against dsDNA antigens.

Patient samples, controls and in separate steps conjugate and mounting medium are applied to the reaction fields of a reagent tray. The BIOCHIP slides are then placed into the recesses of the reagent tray, where all BIOCHIPs of the slide come into contact with the fluids, and the individual reactions commence simultaneously. The fluids are confined to the recessed wells eliminating the need to use a conventional "humidity chamber".

Patient samples are diluted 1:10 in sample buffer 2, $30~\mu \mathrm{l}$ of each diluted patient sample are added to each reaction field of the reagent tray. Reactions are started by fitting the BIOCHIP slides containing the sections from the substrate (Crithidia luciliae smears) into the corresponding

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recesses of the reagent tray and incubated for 30 minutes at room temperature. Specific antibodies attach to the Crithidia luciliae antigens. After incubation the BIOCHIP slides are washed with PBS-Tween to remove unbound antibodies. 25 μl of fluorescein-labelled anti-human globulin are added to each reaction field of a clean reagent tray and the BIOCHIP slides placed into the recesses of the tray. After a 30 minutes incubation at room temperature, the BIOCHIPs are again washed with PBS-Tween to remove any unbound fluorescein-labelled reagent. 10 μl of mounting medium are placed for each reaction field on a cover glass and the BIOCHIP slides, with the BIOCHIPs facing downwards, placed onto the prepared cover glass. Fluorescence is read by EUROPattern fluorescence microscopy.

## M. Performance Characteristics:

1. Analytical performance:

### Modes of comparison:

|  Mode | Imaging | Reading  |
| --- | --- | --- |
|  A | Automated | Automated  |
|  B | Automated | Manual, images on the computer monitor  |
|  C | Manual | Manual, on the microscope  |

Results of the analytical and clinical studies are given separately for the three modes (A, B and C) to demonstrate automated performance using the EUROPattern microscope and software compared to manual performance. For Mode B and C, manufacturer’s predetermined acceptance criteria for performance characteristics were met. Trained operators must confirm all assessments made by the EUROPatter microscope and software.

### Nomenclature:

CLIFT – Crithidia luciliae indirect immunofluorescence test
IIFT – Indirect immunofluorescence test
IF – Immunofluorescence
FI – Fluorescence Intensity

### Titer:

|  Titer | Consideration  |
| --- | --- |
|  Negative | Less than 1:10  |
|  Low positive | 1:10, 1:20, 1:40, 1:80  |
|  Medium positive | 1:160, 1:320, 1:640  |
|  High positive | 1:1280, 1:2560, 1:5120  |

### Interpretation of results:

For a positive result, a distinct, homogeneous, in parts circular fluorescence of the kinetoplast can be identified, the same pattern is found as for the positive control. For negative samples, the kinetoplast shows no staining.

If the positive control shows no specific fluorescence pattern or the negative control shows a clear specific fluorescence, the results are not to be used and the test is to be repeated.

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Qualitative evaluation:

|  Anti-dsDNA reactivity (IgG) | Evaluation  |
| --- | --- |
|  No reaction at 1:10 | Negative. No antibodies against dsDNA detected in the patient sample.  |
|  Positive reaction at 1:10 | Positive. Indication of antibodies to dsDNA in the patient sample.  |

Semi-quantitative evaluation: The endpoint titer is defined as the highest sample dilution factor for which specific fluorescence is identifiable. For a semi-quantitative evaluation all dsDNA positive samples should be reported with endpoint titers, depending on the user established titering protocol. It is stated in the labeling: “For diagnosis, the clinical symptoms of the patient should always be taken into account.”

a. Precision/Reproducibility:

Repeatability/Reproducibility was investigated using a panel of serum samples representing the measurement range (range of patterns and titers) in a laboratory setting at multiple sites. Titer agreement and Percent Positive Agreement (PPA) and Percent Negative Agreements (NPA) were evaluated and summarized. The following samples were tested:

|  Sample | Titer  |
| --- | --- |
|  negative | <10  |
|  low positive | 1:40  |
|  medium positive | 1:320  |
|  high positive | 1:2560  |

Repeatability:

Repeatability was determined by repeated measurements using four samples on five different days with two runs per day and two replicates per run (20 replicates per sample, 80 reads per mode). The IFA: Crithidia luciliae (anti-dsDNA) EUROPattern assays were processed according to the package insert. Each result was reported in three modes (A, B, C). Positive samples were not found negative and vice versa.

Within mode agreement

|  Expected Result | Mode A | Mode B | Mode C  |
| --- | --- | --- | --- |
|   |  % titer agreement (within ±1)  |   |   |
|  Negative | 95 | 100 | 100  |
|  1:40 | 100 | 100 | 100  |
|  1:160 | 85 | 100 | 100  |
|  1:2500 | 95 | 100 | 100  |

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Mode to mode agreement

|  N=80 | Mode A/B | Mode A/C | Mode B/C  |
| --- | --- | --- | --- |
|  % positive agreement (95% CI) | 100 (94.0–100.0) | 100 (94.0–100.0) | 100 (94.0–100.0)  |
|  % negative agreement (95% CI) | 100 (83.2–100.0) | 100 (83.2–100.0) | 100 (83.2–100.0)  |
|  % overall agreement (95% CI) | 100 (95.5–100.0) | 100 (95.5–100.0) | 100 (95.5–100.0)  |
|  % titer agreement (95% CI) | 96.3 (89.4–99.2) | 87.5 (78.2–93.8) | 97.5 (91.3–99.7)  |

Reproducibility:

Reproducibility was evaluated with the same samples as repeatability and was determined by measurements using four samples on five days with two runs per day and two replicates per run, performed at three different sites (60 replicates per sample and 240 reads per mode), at EUROIMMUN's laboratory and two external US clinical laboratories. The IFA: Crithidia luciliae sensitive (anti-dsDNA) EUROPattern assays were processed according to the package insert and evaluated by three different EUROPattern microscope and software systems. Each result was reported in 3 modes (A, B and C). Positive samples were not found negative and vice versa. The results are summarized in the tables below:

Agreement with expected results within mode

|  Sample N=60 | Site | Mode A |   | Mode B |   | Mode C  |   |
| --- | --- | --- | --- | --- | --- | --- | --- |
|   |   |  % positive agreement | % titer agreement within±1 | % Positive agreement | % titer agreement within±1 | % Positive agreement | % titer agreement within±1  |
|  Negative | 1 | 5.0 | 95.0 | 0 | 100 | 0 | 100  |
|   |  2 | 0 | 100 | 0 | 100 | 0 | 100  |
|   |  3 | 0 | 100 | 0 | 100 | 0 | 100  |
|  1:40 | 1 | 100 | 100 | 100 | 100 | 100 | 100  |
|   |  2 | 100 | 100 | 100 | 100 | 100 | 100  |
|   |  3 | 100 | 100 | 100 | 100 | 100 | 100  |
|  1:320 | 1 | 100 | 85.0 | 100 | 90 | 100 | 90  |
|   |  2 | 100 | 95.0 | 100 | 100 | 100 | 100  |
|   |  3 | 100 | 93.8 | 100 | 100 | 100 | 93.8  |
|  1:2560 | 1 | 100 | 95.0 | 100 | 100 | 100 | 100  |
|   |  2 | 100 | 95.0 | 100 | 100 | 100 | 100  |
|   |  3 | 100 | 100 | 100 | 100 | 100 | 100  |

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# Mode to mode agreement at each site

Site 3 had 76 repeats per mode (and not 80) resulting in 236 total reads. The results are summarized in the tables below:

Site 1 mode to mode agreement

|  Site 1; N=236 | A/B | A/C | B/C  |
| --- | --- | --- | --- |
|  % positive agreement (95% CI) | 100 (94.0–100) | 100 (94.0–100) | 100 (94.0–100)  |
|  % negative agreement (95% CI) | 95.0 (75.1–100) | 95.0 (75.1–100) | 100 (83.2–100)  |
|  % overall agreement (95% CI) | 98.8 (93.2–100) | 98.8 (93.2–100) | 100 (95.5–100)  |
|  % titer agreement (95% CI) | 95.0 (87.7–98.6) | 88.8 (79.7–94.7) | 93.8 (95.5–100)  |

Site 2 mode to mode agreement

|  Site 2; N=236 | A/B | A/C | B/C  |
| --- | --- | --- | --- |
|  % positive agreement (95% CI) | 100 (94.0–100) | 100 (94.0–100) | 100 (94.0–100)  |
|  % negative agreement (95% CI) | 100 83.2–100 | 100 (83.2–100) | 100 (83.2–100)  |
|  % overall agreement (95% CI) | 100 (95.5–100) | 100 (87.7-98.6) | 100 (95.5-100)  |
|  % titer agreement (95% CI) | 96.3 (89.4–99.2) | 95.0 (87.7–98.6) | 100 (95.5–100)  |

Site 3 mode to mode agreement

|  Site 3; N=236 | A/B | A/C | B/C  |
| --- | --- | --- | --- |
|  % positive agreement (95% CI) | 100 (93.5–100) | 100 (93.6–100) | 100 (93.6–100)  |
|  % negative agreement (95% CI) | 100 (83.2–100) | 100% (83.2–100) | 100 (83.2–100)  |
|  % overall agreement (95% CI) | 100 (95.3–100) | 100 (95.3–100) | 100 (95.3–100)  |
|  % titer agreement (95% CI) | 97.4 (90.8–99.7) | 100 (95.3–100) | 94.7 (87.1–98.5)  |

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Summary, mode to mode overall agreement at each site

|  N=236 | Overall agreement | A/B | A/C | B/C  |
| --- | --- | --- | --- | --- |
|  Site 1 | % overall agreement (95% CI) | 98.8 (93.2–100) | 98.8 (93.2–100) | 100 (95.5–100)  |
|   |  % titer agreement (95% CI) | 95.0 (87.7–98.6) | 88.8 (79.7–94.7) | 100 (95.5–100)  |
|  Site 2 | % overall agreement (95% CI) | 100 (95.5–100) | 100 (95.5–100) | 100 (95.5–100)  |
|   |  % titer agreement (95% CI) | 96.3 (89.4–99.2) | 95.0 (87.7–98.6) | 100 (95.5–100)  |
|  Site 3 | % overall agreement (95% CI) | 100 (95.3–100) | 100 (95.3–100) | 100 (95.3–100)  |
|   |  % titer agreement (95% CI) | 97.4 (90.8–99.7) | 100 (95.3–100) | 94.7 (87.1–98.5)  |
|  Total | % overall agreement (95% CI) | 99.6 (97.7–100) | 99.6 (97.7–100) | 100 (98.4–100)  |
|   |  % titer agreement (95% CI) | 96.2 (92.9–98.2) | 94.5 (90.8–97.0) | 98.3 (95.7–99.5)  |

Site to site agreement for each mode

Site to site agreement is summarized in the tables below for each mode. Study was evaluated using four samples on five days with two runs per day and two replicates per run, (80 reads per site)

Mode A, site to site comparison

|  Mode A, N=80 | Site 1 vs. 2 | Site 1 vs. 3 | Site 2 vs. 3  |
| --- | --- | --- | --- |
|  % positive agreement (95% CI) | 100 (91.1–100) | 100 (93.6–100) | 100 (94.0–100.0)  |
|  % negative agreement (95% CI) | 95.0 (75.1–99.1) | 95.0 (75.1–99.9) | 100 (83.2–100)  |
|  % overall agreement (95% CI) | 98.8 (93.2–100) | 98.7 (92.9–100) | 100 (95.5–100)  |
|  % titer agreement (95% CI) | 93.8 (86.0–97.9) | 92.1 (83.6–97.0) | 96.1 (88.9–99.2)  |

Mode B, site to site comparison

|  Mode B, N=80 | Site 1 vs. 2 | Site 1 vs. 3 | Site 2 vs. 3  |
| --- | --- | --- | --- |
|  % positive agreement (95% CI) | 100 (94.0–100) | 100 (93.6–100) | 100 (93.6–100)  |
|  % negative agreement (95% CI) | 100.0 (83.2–100) | 100 (83.2–100) | 100 (83.2–100)  |
|  % overall agreement (95% CI) | 100 (95.5–100) | 100 (95.3–100) | 100 (95.3–100)  |
|  % titer agreement (95% CI) | 86.3 (89.4–99.2) | 92.1 83.6–97.0 | 98.7 92.9–100  |

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Mode C, site to site comparison

|  Mode C, N=80 | Site 1 vs. 2 | Site 1 vs. 3 | Site 2 vs. 3  |
| --- | --- | --- | --- |
|  % positive agreement (95% CI) | 100 (94.0–100) | 100 (93.6–100) | 100 (93.6–100.0)  |
|  % negative agreement (95% CI) | 100 (83.2–100) | 100 (83.2–100) | 100 (83.2–100.0)  |
|  % overall agreement (95% CI) | 100 (95.5–100.0) | 100 (95.3–100) | 100 (95.5–100)  |
|  % titer agreement (95% CI) | 95.0 (87.7–98.6) | 86.8 (77.1–93.5) | 96.1 (88.9–99.2)  |

Summary: site to site agreement for each mode

|  Mode N=80 | % agreement | Site 1 vs. 2 | Site 1 vs. 3 | Site 2 vs. 3  |
| --- | --- | --- | --- | --- |
|  Mode A | % overall agreement (95% CI) | 98.8 (93.2–100) | 98.7 (92.9–100) | 100 (95.5–100)  |
|   |  % titer agreement (95% CI) | 93.8 (86.0–97.9) | 92.1 (83.6–97.0) | 96.1 (88.9–99.2)  |
|  Mode B | % overall agreement (95% CI) | 100 (95.5–100) | 100 (95.3–100) | 100 (95.3–100)  |
|   |  % titer agreement (95% CI) | 86.3 (89.4–99.2) | 92.1 (83.6–97.0) | 98.7 (92.9–100)  |
|  Mode C | % overall agreement (95% CI) | 100 (95.5–100.0) | 100 (95.3–100) | 100 (95.5–100)  |
|   |  % titer agreement (95% CI) | 95.0 (87.7–98.6) | 86.8 (77.1–93.5) | 96.1 (88.9–99.2)  |

# Operator to operator comparison

An additional study was performed to evaluate between operator precision by repeated measurments on 5 days with 2 runs per day and 2 replicates per run x 3 samples (for a total of 60 reads). The assays were processed according to the package insert. Each result was reported in 2 modes (B and C). Each mode was performed by 2 different technicians independently. Acceptance criteria were met; positive samples were not found negative and vice versa. The samples used in the study are summarized in the table below:

|  Sample | Titer  |
| --- | --- |
|  Negative | <10  |
|  Low positive | 1:40  |
|  Medium positive | 1:320  |

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Operator to operator agreement with expected results:

|  N=60 | Mode B Two operators | Mode C Two operators  |
| --- | --- | --- |
|  % positive agreement (95% CI) | 100 (91.2–100) | 100 (91.2–100)  |
|  % negative agreement (95% CI) | 100 (83.2–100) | 100 (83.2–100)  |
|  % overall agreement (95% CI) | 100 (94.0–100) | 100 (94.0–100)  |
|  % titer agreement (95% CI) | 100 (94.0–100) | 93.3 (83.8–98.2)  |

## b. Linearity/assay reportable range:

To investigate linearity, three positive samples were serially diluted and assayed with the IFA: Crithidia luciliae sensitive (anti-dsDNA) EUROPattern system. Assays were processed according to the package insert and evaluated by the EUROPattern microscope and software. Individual results per dilution were read visually and reported using a fluorescence intensity scale from “0” (negative) to “5” (high positive). The EUROPattern software creates one result based on all dilutions, it does not allow individual reading of single dilutions. The fluorescence intensities (FI) decreased with dilutions until negative. The results are shown in the table below.

Three samples were used in the study:

|  Sample No | Sample | End titer results estimate per sample  |   |   |
| --- | --- | --- | --- | --- |
|   |   |  Mode A | Mode B | Mode C  |
|  1 | High positive | 1:10240 | 1:5120 | 1:5120  |
|  2 | Medium positive | 1:160 | 1:160 | 1:160  |
|  3 | Low positive | 1:40 | 1:40 | 1:40  |

The dilution results fluorescence intensity (FI) are summarized in the table below:

|  Sample No. | Result per dilution (FI level) Mode C  |   |   |   |   |   |   |   |   |   |
| --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- |
|   |  1:10 | 1:20 | 1:40 | 1:80 | 1:160 | 1:320 | 1:640 | 1:1280 | 1:2560 | 1:5120  |
|  1 | 5 | 5 | 5 | 5 | 5 | 5 | 5 | 3 | 2 | 1  |
|  2 | 5 | 5 | 3 | 2 | 1 | 0 | 0 | 0 | 0 | 0  |
|  3 | 4 | 4 | 2 | 0 | 0 | 0 | 0 | 0 | 0 | 0  |

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

### Traceability:

A recognized standard or reference material for anti-dsDNA antibodies is not available.

### Stability:

Stability studies were conducted in accordance with the international standard DIN EN 13640 / DIN EN ISO 23640: Stability testing of in vitro diagnostic reagents. The stability of the reagents was tested in K172244, the regular IFA: Crithidia luciliae (anti-dsDNA) EUROPattern kit. Only the stability of the different sample buffer (sample buffer 2) was

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investigated for the IFA: Crithidia luciliae sensitive (anti-dsDNA) EUROPattern kit. Real-time stability study was performed using reagents, stored at  $4^{\circ}\mathrm{C}$  and showed stable for up to 36 months. Results met acceptance criteria; compared to those of day. Results were read manually (mode C).

Accelerated stability testing was done on three lots of the sample buffer with one lot of the other reagents (with established stability in K172244 for IFA: Crithidia luciliae (anti-dsDNA), that were stored refrigerated  $4^{\circ}\mathrm{C}$  and at  $37^{\circ}\mathrm{C}$  for 14 days. The reagents are considered to be stable at  $4^{\circ}\mathrm{C}$ . After the end of the storage time, assays were run using a set of two positive and three negative samples. The tests were performed according to the package insert. The deviation in fluorescence intensity level (0-5) did not exceed  $+/- 1$ . For the establishment of the stability claim, a storage time of 14 days at  $+37^{\circ}\mathrm{C}$  equals 18 months at  $+2^{\circ}\mathrm{C}$  to  $+8^{\circ}\mathrm{C}$ . The stability claims are summarized in the table below:

|   | Temperature | Stability claim  |
| --- | --- | --- |
|  Kit Shelf life | +2°C to +8°C | 18 months  |
|  Open kit reagents | +2°C to +8°C | 18 months  |
|  Sample buffer | +2°C to +8°C | 18 months  |
|  Shipping | 37°C | ≤14 days  |

# Control:

Negative and positive controls are included in the kit, ready for use. The positive controls are ready to use human serum that contain anti-dsDNA antibodies that exhibit a distinct, homogeneous, in parts circular fluorescence of the kinetoplast. There is no target titer assigned to the positive controls. The negative control contains ready-to-use autoantibody-negative human sera and should exhibit a negative result. The controls are derived from native samples that are diluted with stabilizing buffer to reach the appropriate ready-to-use concentration. The reactivity of the positive and negative controls is assured by functional and stability testing. EUROIMMUN recommends using the positive and negative controls as stated within the labeling (Instructions for Use).

# d. Detection limit:

Not applicable

# e. Analytical specificity:

# Cross reactivity:

The specificity of the IFA: Crithidia luciliae sensitive (anti-dsDNA) EUROPattern was verified using the ANA reference panel and ANCA (PR3, MPO) positive reference samples obtained from the CDC Centers for Disease Control and Prevention, Atlanta, USA (16 samples). The samples were assayed according to the package insert and evaluated by the EUROPattern microscope and software. Each result was reported in 3 modes (A, B, C). The CDC sample specific for nDNA were positive by the assay; all other samples were negative (for mode B or C); RNP, SP and Ribosomal P-protein were positive for Mode A. The other samples represent other ANA-type antigens such as Scl-70, SS-A, SS-B, Jo-1, etc.

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In addition, the World Health Organization (WHO) Reference Reagent Wo/80 for anti-dsDNA was evaluated by The IFA: Crithidia luciliae sensitive (anti-dsDNA) EUROPattern. The WHO sample specific for dsDNA was positive by the assay.

Additional information on cross-reactivity can be seen in the clinical study differential diagnosis samples that were evaluated for specificity, please see below table in Sensitivity/specificity section, Summary: Sensitivity/Specificity, Correlation with Clinical Diagnosis

Interference: The effect of interfering substances on assay results were tested by spiking normal and positive serum samples with endogenous serum components and drugs. The samples consisted of negative (&lt; 1:10), weak positive, medium positive, and strong positives samples. The spiked samples were processed according to the package insert and evaluated by the EUROPattern microscope and software. Each result was reported in 3 modes (A, B, C). For modes B and C the deviation in titer level did not exceed +/- 1 titer within modes and positive samples were not found negative and vice versa. The summary results are shown in the tables below. No significant interference was observed up to the levels indicated in the table below:

The results are summarized in the table below

|   | Substance | No interference up to  |
| --- | --- | --- |
|  Endogenous | Hemoglobin | 500 mg/dL  |
|   |  Bilirubin | 40 mg/dL  |
|   |  Triglyceride | 2000 mg/dL  |
|   |  RFIgG | 61 U/mL  |
|   |  RF IgM | 1200U/mL  |
|   |  Cyclic Citrullinated Peptide (CCP) | Ratio 8.7 (sample over calibrator)  |
|  Drugs | Cyclophosphamide monohydrate | 14.4 nmol/L  |
|   |  Methotrexate hydrate | 9.1 mg/mL  |
|   |  Azathioprine | 29.0 mg/L  |
|   |  Mycophenolate mofetil | 350 mg/L  |
|   |  Prednisone | 3.0 mg/L  |
|   |  Hydroxychloroquine sulfate | 33.0 μg/mL  |
|   |  Ibuprofen | 5.0 mg/mL  |
|   |  Naproxen sodium | 21.7 mmol/L  |
|   |  Rituximab | 1.1 mg/mL  |
|   |  Belimumab | 1.1 mg/mL  |

f. Assay cut-off:

The recommended starting dilution, above which the result is reported as positive and below which the result is reported as negative, is 1:10. The manufacturer suggests performing two-fold dilutions and also recommends that each laboratory establish its own titering protocol. Assay cut-off of 1:10 was determined from: Renato Tozzoli, MD, Nicola Bizzaro, MD, Elio Tonutti, MD, Danilo Villalta, MD, Danila Bassetti, MD, Fabio Manoni,

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MD, Anna Piazza, MD, Marco Pradella, MD, and Paolo Rizzotti, MD. Guidelines for the Laboratory Use of Autoantibody Tests in the Diagnosis and Monitoring of Autoimmune Rheumatic Diseases. Am J Clin Pathol 2002;117:316 - 324. It states: "In the diagnostic phase, use the highly specific radioimmunologic method (Farr technique) or the IIF assay on Crithidia luciliae at the initial serum dilution of 1:10 to determine anti-dsDNA antibodies."

# 2. Comparison studies:

Method comparison and clinical evaluation of sensitivity and specificity was performed with 364 samples, the samples are detailed in the table below:

|  Panel/disease | Acronym | N (men,women, unknown) | Mean age (range)  |
| --- | --- | --- | --- |
|  Systemic lupus erythematosus | SLE | 98 (20, 78) | 49 (18-85)  |
|  ANCA Associated Vasculitis | AAV | 13 (7, 6) | 65 (31-92)  |
|  Poly-dermatomyositis | PM/DM | 9 (4, 5) | 57 (42-86)  |
|  Systemic sclerosis | SSc | 12 (5, 7) | 55 (39-79)  |
|  Sjögren's syndrome | SS | 20 (0, 20) | 51 (34-88)  |
|  Mixed connective tissue disease | MCTD | 18 (8, 10) | 48 (19-75)  |
|  Rheumatoid arthritis | RA | 49 (11, 38) | 56 (22-87)  |
|  Anti-Phospholipid Syndrome | APS | 30 (20, 10) | 53 (30, 84)  |
|  Drug-Induced Lupus | DIL | 30 (2, 28) | 52 (25, 84)  |
|  Autoimmune hepatitis | AIH | 20 (6, 6, 8) | 56 (33-77)  |
|  Autoimmune thyroid disease | AITD | 16 (6, 10) | 45 (23-63)  |
|  Hepatitis C virus | HCV | 29 (14, 15) | 48 (19-70)  |
|  Clamydia pneumoniae | CP | 10 (9, 1) | 54 (32-73)  |
|  Epstein-Barr virus | EBV | 10 (8, 2) | 44 (27-72)  |
|  Total |  | 364 |   |

# a. Method comparison with predicate device:

Samples were tested with the IFA: Crithidia luciliae sensitive (anti-dsDNA)

EUROPattern assay and the predicate Anti-dsDNA-NcX ELISA (IgG) assay. The results are summarized in the tables below:

Correlation between New Device and Predicate

|  N=364 | EUROIMMUN Anti-dsDNA-NcX ELISA (IgG)  |   |   |   |
| --- | --- | --- | --- | --- |
|   |   |  Positive | Negative | Total  |
|  EUROIMMUN IFA | Positive | 38 | 24 | 62  |
|  Cristedia lucilae (anti-dsDNA) | Negative | 12 | 290 | 302  |
|  EUROPattern Mode C | Total | 50 | 314 | 364  |
|  % positive agreement | 38/50=76.0% | 95%CI: 61.8–86.9  |   |   |
|  % negative agreement | 290/314=92.4% | 95%CI: 88.8–95.0  |   |   |
|  % overall agreement | 324/364=89.0% | 95%CI: 86.6–93.0  |   |   |

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Comparison between modes:

|  Mode | A/B | A/C | B/C  |
| --- | --- | --- | --- |
|  % positive agreement (95% CI) | 65/66=98.5% (91.8–100) | 62/62=100% (94.2–100) | 61/62=98.4% (91.3–100)  |
|  % negative agreement (95% CI) | 272/298=91.3% (87.5–94.2) | 273/302=90.4% (86.5–93.5) | 297/302=98.3% (96.2–99.5)  |
|  % overall agreement (95% CI) | 337/364=92.6% (89.4–95.1) | 337/364=92.0% (88.8–94.6) | 358/364=98.4% (96.4–99.4)  |
|  Titer agreement ±1 titer | 319/364=87.6% (83.8–90.8) | 319/364=87.6% (83.8–90.8) | 357/364=98.1% (96.1–99.2)  |

b. Matrix comparison:

Not applicable, any serum is used in the assay.

3. Clinical studies:

Clinical studies were performed at three sites (two external US clinical laboratories and EUROIMMUN's laboratory). In total 364 clinically characterized samples, diagnosed according to the American College of Rheumatology (ACR) or other appropriate classification criteria, were investigated for anti-dsDNA antibodies. Samples were assayed with the IFA: Crithidia luciliae sensitive (anti-dsDNA) EUROPattern according to the package insert and evaluated by the EUROPattern microscope and software. Each result was reported in 3 modes (A, B, C). The overall sensitivity (Se) and specificity (Sp) of the system was evaluated,  $95\%$  C.I. are calculated by the exact method.

a. Clinical Sensitivity/Specificity:

Sensitivity and specificity was evaluated for each mode at each site and at the three sites combined (364 x 3 sites = 1092 total samples; 98 positive x 3 sites = 294; 266 negative x 3 sites = 798)

Correlation to clinical diagnosis for each mode at the three sites

|  N=1092 | Clinical diagnosis  |   |   |
| --- | --- | --- | --- |
|   |   |  Positive/per total SLE | Negative/per total non-SLE  |
|  EUROIMMUN IFA Criedia lucilae (anti-dsDNA) EUROPattern | Mode A | 143/294 | 722/798  |
|   |  Mode B | 140/294 | 753/798  |
|   |  Mode C | 135/294 | 756/798  |

Summary: clinical overall agreement for all three sites for each mode

|  Overall Sensitivity/specificity (95% CI) | Method A | Method B | Method C  |
| --- | --- | --- | --- |
|  SensitivityN=294 | 143/294=48.6%(42.8–54.5) | 140/294=47.6%(41.8–53.5) | 135/294=45.9%(40.1–51.8)  |
|  SpecificityN=798 | 722/798=90.5%(88.2–92.4) | 753/798=94.4%(92.5–95.9) | 756/798=94.7%(93.0–96.2)  |

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Correlation with SLE clinical diagnosis in each mode at each site

|  Sensitivity (95% CI) N=98 | Mode A | Mode B | Mode C  |
| --- | --- | --- | --- |
|  Site 1 | 53/98=54.1% (43.7–64.2) | 47/98=48.0% (37.8–58.3) | 45/98=45.9% (35.8–56.3)  |
|  Site 2 | 45/98=45.9% (35.8–56.3) | 44/98=44.9% (34.8–55.3) | 43/98=43.9% (33.9–54.3)  |
|  Site 3 | 45/98=45.9% (35.8–56.3) | 49/98=50.0% (39.7–60.3) | 47/98=48.0% (37.8–58.3)  |

Correlation with differential diagnosis in each mode at each site

|  Specificity (95% CI) N=266 | Mode A | Mode B | Mode C  |
| --- | --- | --- | --- |
|  Site 1 | 288/266=85.7% (80.9–89.7) | 247/266=96.2% (89.1–95.6) | 249/266=93.6% (90.0–96.2)  |
|  Site 2 | 248/266=93.2% (89.5–95.9) | 254/266=95.5% (92.3–97.6) | 254/266=95.5% (92.3–97.6)  |
|  Site 3 | 246/266=92.5% (88.6–95.3) | 252/266=94.7% (91.3–97.1) | 253/266=95.1% (91.8–97.4)  |

Summary: sensitivity/specificity, correlation with clinical diagnosis by disease

|  Disease | N | Site 1 |   |   | Site 2 |   |   | Site 3  |   |   |
| --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- |
|   |   |  Mode A | Mode B | Mode C | Mode A | Mode B | Mode C | Mode A | Mode B | Mode C  |
|   |   |  Sensitivity: SLE  |   |   |   |   |   |   |   |   |
|  SLE | 98 | 54.1 | 48.0 | 45.9 | 45.9 | 44.9 | 43.9 | 45.9 | 50.0 | 48.0  |
|   |  | Specificity: Non-SLE  |   |   |   |   |   |   |   |   |
|  AAV | 13 | 92.3 | 92.3 | 100 | 100 | 100 | 100 | 92.3 | 100 | 100  |
|  PM/DM | 9 | 100 | 100 | 100 | 100 | 100 | 100 | 88.9 | 88.9 | 88.9  |
|  SSc | 12 | 91.7 | 100 | 100 | 91.7 | 91.7 | 91.7 | 91.7 | 91.7 | 100  |
|  SS | 20 | 90.0 | 100 | 95.0 | 90.0 | 100 | 100 | 95.0 | 100 | 100  |
|  MCTD | 18 | 100 | 100 | 100 | 100 | 100 | 100 | 100 | 100 | 100  |
|  RA | 49 | 93.9 | 98.0 | 98.0 | 95.9 | 89.5 | 95.9 | 93.9 | 98.0 | 98.0  |
|  APS | 30 | 93.3 | 100 | 100 | 100 | 100 | 100 | 100 | 100 | 100  |
|  DIL | 30 | 53.3 | 76.7 | 76.7 | 76.7 | 83.3 | 83.3 | 80.0 | 80.0 | 80.0  |
|  AIH | 20 | 80.0 | 85.0 | 85.0 | 90.0 | 90.0 | 90.0 | 90.0 | 90.0 | 90.0  |
|  AITD | 16 | 81.3 | 81.3 | 81.3 | 93.8 | 93.8 | 93.8 | 93.8 | 93.8 | 93.8  |
|  HCV | 29 | 82.8 | 93.1 | 93.1 | 93.1 | 96.6 | 96.6 | 93.1 | 93.1 | 93.1  |
|  CP | 10 | 80.0 | 90.0 | 100 | 100 | 100 | 100 | 100 | 100 | 100  |
|  EBV | 10 | 90.0 | 90.0 | 100 | 90.0 | 100 | 100 | 80.0 | 100 | 100  |
|  Total | 266 | 85.7 | 92.9 | 93.6 | 93.2 | 95.5 | 95.5 | 92.5 | 94.7 | 95.1  |

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b. Clinical specificity:
See Clinical Sensitivity

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

4. Clinical cut-off:
See assay cut-off

5. Expected values/Reference range:
Positive dsDNA results detected by IFA are not commonly seen in normal populations and the expected value in the normal population is “negative” at a 1:10 starting dilution. Anti-dsDNA antibodies were analyzed with the IFA: Crithidia luciliae sensitive (anti-dsDNA) EUROPattern system using a panel of 98 sera from normal U.S. healthy adult blood donors of mixed age and gender (35 men, 63 women, mean age 37 years, range 19-50 years). The samples were assayed according to the package insert and evaluated by the EUROPattern microscope and software. Each result was reported in 3 modes (A, B, C). At the cut-off dilution of 1:10, the prevalence was found to be 2% for mode B or C.

The manufacturer recommends that each laboratory establish its own normal range based on the population and equipment used.

N. Instrument Name:
EUROPattern

O. System Descriptions:

1. Modes of Operation:

Does the applicant’s device contain the ability to transmit data to a computer, webserver, or mobile device?
Yes ☐ X or No ☐

Does the applicant’s device transmit data to a computer, webserver, or mobile device using wireless transmission?
Yes ☐ or No ☐ X

2. Software:

FDA has reviewed applicant’s Hazard Analysis and software development processes for this line of product types:
Yes ☐ X or No ☐

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3. Specimen Identification:
Human serum

4. Specimen Sampling and Handling:
Starting dilution is 1:10

5. Calibration:
Samples are evaluated on the microscope, there is no calibrator in the assay.

6. Quality Control:
Positive and negative control are part of the assay kit

P. Other Supportive Instrument Performance Characteristics Data Not Covered In The "Performance Characteristics" Section above:
N/A

Q. Proposed Labeling:
The labeling is sufficient and it satisfies the requirements of 21 CFR Part 809.10.

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

18

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**Source:** [https://fda.innolitics.com/submissions/IM/subpart-f%E2%80%94immunological-test-systems/KTL/K172252](https://fda.innolitics.com/submissions/IM/subpart-f%E2%80%94immunological-test-systems/KTL/K172252)

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