EliA Scl-70s is intended for the in vitro semi-quantitative measurement of IgG antibodies directed to Scl-70 in human serum and plasma (heparin, EDTA) as an aid in the clinical diagnosis of scleroderma (diffuse form) in conjunction with other laboratory and clinical findings. EliA Scl-70s uses the EliA IgG method on the instrument Phadia 100.

Device Description

The Phadia EliA immunodiagnostic system is automated system for immunodiagnostic testing. The EliA reagents are available as modular packages, each purchased separately. All packages except the positive and negative controls are required to carry out an EliA Scl-70° test.

AI/ML Overview

The acceptance criteria and study detailed in the provided document pertain to the EliA™ Scl-70S Immunoassay, an in vitro semi-quantitative test for IgG antibodies directed to Scl-70.

Here's a breakdown of the requested information:

1. Table of Acceptance Criteria and Reported Device Performance

The document does not explicitly state pre-defined acceptance criteria for the clinical performance (sensitivity and specificity). However, it reports the performance of the EliA Scl-70S based on its clinical study. For analytical performance, the acceptance criteria are implied by the reported results meeting generally accepted ranges for such assays.

Performance Characteristic	Acceptance Criteria (Implied)	Reported Device Performance (EliA Scl-70S)
Analytical Performance
Precision/Reproducibility
Phadia 100 (Total %CV)	Low (<10%)	3.55% (8.8 EliA U/ml), 4.01% (30.2 EliA U/ml), 6.03% (193.0 EliA U/ml)
Phadia 250 (Total %CV)	Low (<10%)	3.33% (7.5 EliA U/ml), 3.53% (28.2 EliA U/ml), 5.40% (200.1 EliA U/ml)
Linearity (R²)	Close to 1.00	1.00 across various dilution ranges (both instruments)
Hook Effect	No hook effect up to a certain concentration	No hook effect observed up to 14 times above upper limit of measuring range
Limit of Detection (LoD)	Defined value	0.6 EliA U/mL (for both instruments)
Analytical Specificity (Interference)	No significant interference observed	Ratio of blank/spiked sample 0.94 – 1.09 with various interferents (Bilirubin, Hemoglobin, Chyle, Rheumatoid factor)
Carry-over	Negligible effect	Observed carry-over effect is negligible without any influence on assay results
Method Comparison with Predicate Device (Technical Agreement)	High agreement with predicate	Equivocal results evaluated as negative:Positive Percent Agreement: 89.5% (95% CI: 77.8 – 95.6%)Negative Percent Agreement: 97.2% (95% CI: 94.5 – 98.6%)Total Percent Agreement: 95.9% (95% CI: 93.3 – 97.7%)Equivocal results evaluated as positive:Positive Percent Agreement: 93.0% (95% CI: 82.2 – 97.8%)Negative Percent Agreement: 93.4% (95% CI: 89.9 - 95.8%)Total Percent Agreement: 93.3% (95% CI: 90.2 - 95.5%)
Matrix Comparison (Serum vs Plasma)	Slopes close to 1, intercepts close to 0, high R²	Serum vs Heparin plasma: Slope 1.05 (1.03-1.07), Intercept -0.08 (-0.24 to +0.17), R² 1.00Serum vs EDTA plasma: Slope 1.00 (0.99-1.02), Intercept -0.05 (-0.29 to +0.21), R² 1.00
Instrument Comparison (Phadia 250)	Slopes close to 1, intercepts close to 0	Estimate: Intercept -0.07, Slope 0.96 (for regression analysis)
Clinical Performance (Equivocal results evaluated as negative)	Desirable sensitivity and specificity for aid in diagnosis	Sensitivity: 30.7% (95% CI: 22.1% - 40.8%)Specificity: 98.7% (95% CI: 96.0% - 99.7%)

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

Sample Size for Analytical Performance Tests:
- Precision/Reproducibility: 3 samples for Phadia 250, 1 batch for Phadia 100.
  - Phadia 100: 84 replicate determinations per sample (21 runs: 3 instruments x 7 runs each, over 7 days).
  - Phadia 250: 252 replicate determinations per sample (21 runs: 3 instruments x 7 runs each, over 7 days).
- Linearity: 4 patient serum samples.
- Hook Effect: One high positive serum sample.
- Detection Limit (LoB/LoD): 6 blood donors. Each measured in 12 replicates in each of 6 runs on 6 different days (432 replicates per sample).
- Endogenous Interference: 5 serum samples (2 negative, 2 around cut-off, 1 positive).
Sample Size for Comparison Studies:
- Method Comparison with Predicate Device: 390 serum samples.
  - Collected from patients with Scleroderma (SSc, n = 101), CREST (n = 33), MCTD (n = 37), SLE (n = 34), Sjögren's syndrome (SS, n = 26), Poly/Dermatomyositis (PM/DM, n = 5), Rheumatoid arthritis (RA, n = 30), various cancers (n = 20), various bacterial infections (n = 24), various viral infections (n = 26), and technical samples positive or equivocal for EliA Scl-70° (n = 54).
- Matrix Comparison: 50 patients (serum, lithium heparin plasma, EDTA plasma collected from each).
- Instrument Comparison: 24 positive, 8 equivocal, and 4 negative samples (total 36).
Sample Size for Clinical Studies:
- Clinical Sensitivity and Specificity: 336 clinically defined sera.
  - Diagnostic groups: Scleroderma (SSc, n=101) and Controls (n=235) which include other connective tissue diseases, bacterial/viral infections, cancer, and rheumatoid arthritis.
- Assay Cut-off / Expected values: 400 apparently healthy blood donor samples from Caucasian individuals (equally distributed by sex and age).
Data Provenance: The document does not explicitly state the country of origin for the patient or blood donor samples. It is implied to be retrospective as samples were "collected from patients with diagnosis of..." for the comparison and clinical studies. For the assay cut-off, "400 apparently healthy blood donor samples from Caucasian individuals" were used.

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

The document does not specify the number of experts used or their qualifications to establish the ground truth for the clinical diagnosis of scleroderma (SSc) or other conditions in the test set. It states "clinically defined sera from patients with scleroderma or other connective tissue diseases, bacterial or viral infections, cancer or rheumatoid arthritis," implying that the diagnosis was based on standard clinical practice rather than a dedicated expert panel for this study.

4. Adjudication Method for the Test Set

The document does not describe an adjudication method for establishing ground truth diagnoses for the clinical test set. Clinical findings are referenced as the basis for diagnosis ("clinically defined sera"), but no specific multi-reader adjudication process is mentioned for the study itself.

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

No, a Multi-Reader Multi-Case (MRMC) comparative effectiveness study was not performed or reported in this document. The device is a laboratory immunoassay, and its performance is evaluated against a predicate device and clinical diagnoses, not as an aid to human readers in interpreting images or other data.

6. Standalone (i.e., algorithm only without human-in-the-loop performance) Study

Yes, the study is inherently a standalone performance evaluation of the immunoassay itself. The reported performance characteristics (precision, linearity, detection limits, clinical sensitivity, specificity) reflect the algorithm's (immunoassay's) ability to detect the target antibodies in patient samples without direct human-in-the-loop interpretation once the sample is loaded into the automated Phadia instruments. The results are quantitative (EliA U/mL) leading to a categorical output (negative, equivocal, positive) based on predefined cut-offs.

7. The Type of Ground Truth Used

The ground truth used for the clinical study was clinical diagnosis. The document states "336 clinically defined sera from patients with scleroderma or other connective tissue diseases, bacterial or viral infections, cancer or rheumatoid arthritis." This implies that the ground truth was established by medical professionals through standard diagnostic procedures (e.g., patient history, physical examination, other laboratory tests, imaging, etc.) to arrive at a clinical diagnosis.

For the method comparison, the ground truth was essentially the results from the predicate device (INOVA QuantaLite™ Scl-70 ELISA), against which the new device's technical agreement was measured.

8. The Sample Size for the Training Set

The document does not explicitly describe a separate training set for the device's development or a study related to training a machine learning model. This immunoassay device is a chemical/biological test system with predefined reagents and a specific detection methodology, not a machine learning algorithm that typically requires a distinct training phase with a labelled dataset. Its calibration employs calibrators and controls.

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

As there is no explicit training set discussed in the context of machine learning or algorithm training, this question is not directly applicable. For the immunoassay, the "ground truth" for calibration and controls would be established by the manufacturer through rigorous characterization and standardization of the calibrator and control materials, often traceable to international reference preparations (e.g., WHO standards for IgG). The document mentions that "New batches of IgG calibrators are compared to a secondary standard (standardized with the IRP) or the IRP directly and adjusted accordingly to meet the correct concentration."

Summary

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Food and Drug Administration 10903 New Hampshire Avenue Document Control Center - WO66-G609 Silver Spring, MD 20993-0002

October 30, 2014

PHADIA US, INC. C/O MR. MARTIN ROBERT MANN SENIOR REGULATORY AFFAIRS MANAGER 4169 COMMERCIAL AVENUE PORTAGE, MI 49002

Re: K140493

Trade/Device Name: EliA™ Scl-708 Immunoassay Regulation Number: 21 CFR §866.5100 Regulation Name: Antinuclear Antibody Immunological Test System Regulatory Class: Class II Product Code: LJM Dated: September 24, 2014 Received: September 26, 2014

Dear Mr. Mann:

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

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If you desire specific advice for your device on our labeling regulation (21 CFR Part 801), please contact the Division of Industry and Consumer Education at its toll-free number (800) 638-2041 or (301) 796-7100 or at its Internet address

http://www.fda.gov/MedicalDevices/Resourcesfor You/Industry/default.htm. Also, please note the regulation entitled. "Misbranding by reference to premarket notification" (21CFR 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 the CDRH's Office of Surveillance and Biometrics/Division of Postmarket Surveillance.

You may obtain other general information on your responsibilities under the Act from the Division of Industry and Consumer Education at its toll-free number (800) 638-2041 or (301) 796-7100 or at its Internet address

http://www.fda.gov/MedicalDevices/ResourcesforYou/Industry/default.htm.

Sincerely vours.

Maria M. Chan -S

Maria M. Chan, Ph.D. 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) K140493

Device Name EliA™ Scl-70s

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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510k Summary long EliA Scl-70S D.1

510(k) SUBSTANTIAL EQUIVALENCE DETERMINATION SUMMARY

A. 510(k) Number: K140493
B. Purpose for Submission: New devices
C. Measurand: lgG Antibodies to Scl-70
D. Type of Test: Semi-quantitative immunofluorescence assay
E. Applicant: Phadia US Inc.
F. Proprietary and Established Names: EliA™ Scl-708 Well
G. Regulatory Information: 1. Regulation section: 21 CFR §866.5100, Antinuclear antibody immunological test system
- 2. Classification: Class II (Assay)
- 1. Product code: LJM Antinuclear Antibody (Enzyme-Labeled), Antigen, Controls|
- 1. Panel: Immunology

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H. Intended Use:

1. Intended use(s):

EliA Scl-70° is intended for the in vitro semi-quantitative measurement of IgG antibodies directed to Scl-70 in human serum and plasma (heparin, EDTA) as an aid in the clinical diagnosis of scleroderma (diffuse form) in conjunction with other laboratory and clinical findings. EliA Scl-70 uses the EliA IgG method on the instrument Phadia 100.

1. Indication(s) for use: Same as intended use
1. Special conditions for use statement(s): For prescription use only
1. Special instrument requirements: Phadia® 100, Phadia® 250

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l. Device Description:

EliA Scl-70 Wells (14-5637-01) are coated with a human recombinant Scl-70 protein -4 carriers, (12 wells each), ready to use
The method-specific reagents on Phadia 250 are identical; they are only filled in different containers.

Needed reagents for Phadia 100:

EliA Sample Diluent, method-specific, PBS containing BSA, detergent and 0.095% sodium azide . - ready to use: (83-1003-01)
EliA IgG Conjugate, ß-galactosidase labeled anti-IgG (mouse monoclonal antibodies) in PBS containing BSA, detergent and 0.06% sodium azide - ready to use; (83-1002-01, 83-1005-01)
EliA ANA Positive Control 100: containing IgG antibodies to dsDNA, RNP, Sm, Ro, La, Scl-70, . CENP and Jo-1, 6 single-use vials, 0.3 mL each, ready to use; (83-1038-01)
EliA IgG/IgM/IgA Negative Control 100: containing normal human serum from healthy donors -. 6 single-use vials, 0.3 mL each, ready to use; (83-1042-01)
EliA IgG Calibrators, human IgG in PBS (0, 4, 10, 20, 100, 600 µg/L) 6 single-use vials, 0.3 mL . each, ready to use; (83-1000-01)
EliA IgG Curve Control, human IgG (20 µg/L) in PBS, 6 single-use vials, 0.3 mL each, ready to . use; (83-1001-01)
EliA IgG Calibrator Wells are coated with mouse monoclonal antibodies 4 carriers. (12 wells . each), ready to use; (14-5509-01)

Needed reagents for Phadia 250:

EliA Sample Diluent, method-specific, PBS containing BSA, detergent and 0.095% sodium azide . - ready to use; (83-1023-01)
EliA IgG Conjugate, ß-galactosidase labeled anti-IgG (mouse monoclonal antibodies) in PBS containing BSA, detergent and 0.06% sodium azide - ready to use; (83-1017-01, 83-1018-01)
. EliA ANA Positive Control 250: containing IgG antibodies to dsDNA, RNP, Sm, Ro, La, Scl-70, CENP and Jo-1, 6 single-use vials, 0.3 mL each, ready to use; (83-1033-01)
EliA IgG/IgM/IgA Negative Control 250: containing normal human serum from healthy donors -. 6 single-use vials, 0.3 mL each, ready to use; (83-1037-01)
EliA IgG Calibrator Strips, human IgG in PBS (0, 4, 10, 20, 100, 600 µg/L) 6 single-use vials, . 0.3 mL each, ready to use; (83-1015-01)
EliA IgG Curve Control Strips, human IgG (20 µg/L) in PBS, 6 single-use vials, 0.3 mL each, . ready to use; (83-1016-01)
EliA IgG Calibrator Wells are coated with mouse monoclonal antibodies 4 carriers, (12 wells each), ready to use; (14-5509-01)

The Phadia EliA immunodiagnostic system is automated system for immunodiagnostic testing. The EliA reagents are available as modular packages, each purchased separately. All packages except the positive and neqative controls are required to carry out an EliA Scl-70° test.

J. Substantial Equivalence Information:

1. Predicate device name(s) and 510(k) number(s): QuantaLite™ Scl-70 ELISA, INOVÀ K924988

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2. Comparison with predicate:

Similarities to predicate device

Feature	Predicate Device	New Device
	INOVA QuantaLite Scl-70 ELISA	EliA Scl-70S
Intended Use	QUANTALite Scl-70 is an en-zyme-linked immunosorbent as-say (ELISA) for the semi-quantitative detection of Scl-70antibodies in human serum. Thepresence of Scl-70 antibodiescan be used in conjunction withclinical findings and other labora-tory tests to aid in the diagnosisof scleroderma.	EliA Scl-70S is intended for the invitro semi-quantitative measure-ment of IgG antibodies directedto Scl-70 in human serum andplasma (heparin, EDTA) as anaid in the clinical diagnosis ofscleroderma (diffuse form) inconjunction with other laboratoryand clinical findings. EliA Scl-70Suses the EliA IgG method on theinstruments Phadia 100 or Pha-dia 250.
Internal Controls	Low Positive, High Positive andNegative Controlincluded in the kit	Positive and negative Controlprovided with the EliA ANA Posi-tive Control 100 / 250 and EliAIgG/IgM/IgA Negative Control100 / 250, respectively
Assay Type	ELISA
Type of test	Semi-quantitative
Solid Phase	Microwells

Differences to predicate device

Differences to predicate device
Feature	Predicate DeviceINOVA QuantaLite Scl-70 ELISA	New DeviceEliA Scl-70S
Antigen used	purified Scl-70 antigen	Human recombinant Scl-70 pro-tein
Instrumentation	ELISA-Reader needed	Phadia 100, Phadia 250 are fullyautomated immunoassay ana-lyzers
Reaction tempera-ture	Room temperature, 20-26°C	37°C controlled
Incubation times	High positive, low positive andnegative Controls, diluted patientsamples: 30 min.Conjugate: 30 min.Substrate: 30 min (in dark).	Diluted patient samples: 30 min.Conjugate: 28 min.Development Solution: 39 min.
Detection antibody(conjugate)	IgG conjugate: anti-human IgGhorse radish peroxidase (goat)	IgG conjugate: anti-human IgGβ-Galactosidase (mouse mono-clonal antibodies)
Signal	Optical density (at 450nm)	Fluorescence
Calibration	1-point Calibration	Total IgG Calibration
Calibrators	n.a.	6 vials of human IgG at concen-trations of0 - 4 - 10 - 20 - 100 - 600 µg/l
Calibration curve	n.a.	Option to store curve for up to 28days and run curve controls ineach assay for calibration
Concept	All reagents in a single kit	Modular reagents concept(test-method specific and general

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Feature	Predicate DeviceINOVA QuantaLite Scl-70 ELISA	New DeviceEliA Scl-70Sreagents)
Sample Dilution	1:101	1:100
Reported Unit	Units	EliA U/ml
Cut-off	< 20 Units negative20-39 Units weak positive40-80 Units moderate positive> 80 Units strong positive	< 7.0 EliA U/ml negative7.0-10.0 EliA U/ml equivocal> 10.0 EliA U/ml positive
Substrate	TMB Chromogen	Development Solution 0.01 %4-Methylumbelliferyl-β-D-galactoside & <0.0010% pre-servative (mixture of 5-chloro-2-methyl-2H-isothiazol-3-one [ECno. 247-500-7] and 2-methyl-2H-isothiazol-3-one [EC no. 220-239-6] (3:1))

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

CLSI EP17-A, Protocols for Determination of Limits of Detection and Limits of Quantification, Approved Guideline.

L. Test Principle:

The EliA Scl-708 Wells are coated with human recombinant Scl-70 protein. If present in the patient's specimen, antibodies to Scl-70 bind to their specific antigen. After washing away non-bound antibodies, enzyme-labeled antibodies against human IgG antibodies (EliA IgG Conjugate) are added to form an antibody-conjugate complex. After incubation, non-bound conjugate is washed away and the bound complex is incubated with a Development Solution. After stopping the reaction, the fluorescence in the reaction mixture is measured. The assay directly measures the amount of antibody of interest bound to the antigen coating the EliA well, therefore the higher the value of fluorescent signal detected by the instrument, the higher the amount of antibody bound and detected in the sample tested. To evaluate test results, the response for patient samples is compared directly to the response for calibrators.

M. Performance Characteristics (if/when applicable):

Analytical performance:

Precision/Reproducibility: a.

To determine the precision of the assays on Phadia 250 instruments, the variability was assessed on 3 samples. One batch was used to determine the precision of the assays on Phadia 100.

Three batches were used to determine the precision of the assays on Phadia 250 (equal to 252 replicate determinations per sample). The results are summarized in the tables below:

To determine the precision of the assays, the variability was assessed in a study with a total of 21 runs (3 instruments x 7 runs each) over 7 days performed on samples with 84 replicate determinations per sample with a calibration curve in each run.

Mean valueEliA U/ml	Intra-RunCV%	Inter-RunCV%	Total%CV
8.8	2.89	2.06	3.55
30.2	3.21	2.41	4.01
193.0	4.90	3.51	6.03

EliA Scl-708 on Phadia 100 (n=84)

To determine the precision of the assay, the variability was assessed in a study with a total of 21 runs (3 instruments x 7 runs each) over 7 days performed on samples with 252 replicate determinations per sample with a calibration curve in each run.

Mean valueEliA U/ml	Intra RunCV%	Inter RunCV%	Total%CV	Lot to LotCV%
7.5	3.08	1.25	3.33	1.07
28.2	2.71	2.26	3.53	2.01
200.1	4.46	3.05	5.40	5.27

EliA Scl-70 on Phadia 250 (n = 252)

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Linearity/assay reportable range: b.

Four patient serum samples were diluted in sample diluent and tested with one batch of EliA Scl-70° with one set of system reagents on Phadia 250. The ratios of observed/expected values and least square linear regressions were calculated. The results are summarized below:

EliA Scl-708 on Phadia 100

Dilution range (EliA U/ml)	Slope	Intercept	R2
172.3 - 1.8	1.112	-0.902	1.00
185.5 - 2.1	0.995	0.151	1.00
192.0 - 2.1	1.062	0.300	1.00
28.1 - 0.1	0.960	-0.142	1.00
297.1 - 2.9	1.101	-1.134	1.00

EliA Scl-708 on Phadia 250

Dilution range(EliA U/ml)	Slope	Intercept	R2
207.7 - 1.9	0.961	-0.399	1.00
159.9 - 1.4	1.088	0.655	1.00
193.6 - 2.1	1.062	0.047	1.00
24.3 - 0.2	1.041	0.040	1.00
298.9 - 3.1	1.088	0.653	1.00

The claimed linear range for EliA Scl-70° is 0.6 - 297.1 EliA U/ml (0.6 is the limit of detection).

HookEffect/OvertheRangeResults:

Hook effect was investigated by using a serum sample above the highest calibration point. A high positive sample was diluted and the dilutions were measured in four replicates and compared to Cal-600 (600 µg/L).

No hook effect was observed when analyzing a high positive sample that had a concentration up to 14 times above the upper limit of the technical measuring range.

Results above the upper limit of the measuring range are reported as "above". No recommendations are made for dilution of samples outside measuring range in the Package Insert.

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

The IgG calibrators are traceable (via unbroken chain of calibrations) to the International Reference Preparation (IRP) 67/86 of Human Serum Immunoglobulins A. G and M from WHO. New batches of IgG calibrators are compared to a secondary standard (standardized with the IRP) or the IRP directly and adjusted accordingly to meet the correct concentration.

The instrument measures specific IgG concentrations in ug/l. By using a conversion factor given as bar code on the EliA well, the results are automatically converted to EliA U/mL for EliA Scl-70°.

Stability:

The EliA IgG system reagents EliA ANA Positive Control and the EliA IgG/IgM/IgA Negative Control are already FDA cleared under K063775, K072393, and K091845, respectively.

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Shelf life:

Accelerated study was done to determine the shelf life EliA Scl-70 Wells and was determined to be 18 months at 2-8°C. The real time stability test confirms the determined shelf life.

On-board stability:

The on-board stability EliA Scl-70 carriers was tested over 4 weeks using 3 positive and 2 negative samples only on the Phadia 250 instrument since for Phadia 100 the reagents are stored off the instrument and are only loaded as needed for an assay. The on-board stability for the Phadia 250 instrument was determined to be 28 days at 2-8°C.

Open Stability:

Accelerated study was done to determine the shelf life EliA Scl-70° Wells after first opening of the foil bag containing the EliA Scl-70° Wells. It was determined to be 9 months at 2-8°C. The real time stability test confirms the determined shelf life.

d. Detection limit:

The limit of blank (LoB) and limit of detection (LoD) studies were done on both Phadia 100 and Phadia 250. Six blood donors with low/negative antibody concentration were measured in twelve replicates in each of six runs on six different days (12 replicates x 6 runs x 6 days = 432 replicates per sample). The six runs for each system, Phadia 100 and Phadia 250, were spread over two instruments. The results are summarized in the tables below:

EliA Scl-70S (EliA U/mL)	LoB	LoD
Phadia 100	0.25	0.36
Phadia 250	0.31	0.52

It was decided to use a single LoD of 0.6 U/mL for both instruments.

Analytical specificity: e.

Endogenous Interference:

A study was run to investigate whether high concentrations of potentially interfering substances in serum, like bilirubin, hemoglobin, chyle and rheumatoid factor adversely affect the results of the new device. Five serum samples were prediluted in EliA Sample Diluent and spiked with the different interfering substances or their respective blank solutions, and analyzed in triplicates. The runs were repeated twice. One batch of EliA antigen wells and one batch of system reagents were used throughout the studies.

Two negative, two samples with concentrations around the cut-off and one positive sample were tested. The ratio of blank/spiked sample was 0.94 – 1.09. No interference was observed up to the concentrations listed in the table below:

Potential Interfering Compound	Concentration in undiluted sample
Bilirubin F	19.2 mg/dl
Bilirubin C	20.1 mg/dl
Hemoglobin	496 mg/dl
Chyle (ClinOleic)	1%
Rheumatoid factor	500 IU/ml

Carry-over:

Carry over is a technical phenomenon that is due to the instrumentation. The instrumentation is identical for all EliA tests (Phadia instruments). Also the basic circumstances of the test are identical between EliA Ro and the newly submitted EliA test (same source of test material, same reagents, same magnitude of specific antibody concentration etc.). Thus, results obtained with one EliA test can be transferred to any other EliA serum test.

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A study was carried out on a Phadia 250 instrument using the test EliA Ro, cleared under K082759. A serum sample was diluted 1:2 and 1:20 using instrument dilution and manual dilution. A lower dilution factor than the default one (1:100) was chosen to challenge the system. Only a few RUs difference compared to the reference pipetting could be seen, which is too low to be expressed in EliA U/ml.

The observed carry over effect is therefore negligible without any influence to assay results.

f. Assay cut-off:

A study was done on 400 apparently healthy blood donor samples from Caucasian individuals equally distributed by sex and age in order to evaluate expected values in the normal population and to confirm the defined cut-off. The samples were measured on the Phadia 250 instrument for EliA Scl-70°. The 99th percentile of the 400 samples were calculated and taken into account for setting of the cut-off. The following values were selected for the cut-off:

EliA Scl-708
<7 EliA U/mL	Negative
7 — 10 EliA U/mL	Equivocal
>10 EliA U/mL	Positive

In case of equivocal results, it is recommended to retest the patient after 8-12 weeks.

2. Comparison studies:

Method comparison with predicate device: a.

A total of 390 serum samples were collected from patients with diagnosis of Scleroderma (SSc. n = 101), limited cutaneous form of systemic sclerosis (CREST, n = 33), Mixed connective tissue disease (MCTD, n = 37), Systemic lupus erythematodes (SLE, n = 34), Sjögren's syndrome (SS, n = 26), Poly/Dermatomyositis (PM/DM, n = 5), Rheumatoid arthritis (RA, n = 30), various cancers (n = 20), various bacterial infections (n = 24), various viral infections (n = 26), technical samples positive or equivocal for EliA Scl-70° (n = 54).

Samples were analyzed with the EliA Scl-70° and INOVA QuantaLite™ Scl-70 ELISA assays. The test was run in single determination and values outside the linear measuring range (n=16) were excluded from statistical analyses for the technical agreement. The results are summarized in the tables below:

QuantaLite Scl-70 ELISA (Units)
	Positive >20	Negative <20	Total
EliA Scl-70S(EliA U/mL)	Positive >10	51	9	60
	Negative ≤10	6	308	314
	Total	57	317	374
Positive percent agreement: 89.5% (95% CI: 77.8 – 95.6%)
Negative percent agreement: 97.2% (95% CI: 94.5 – 98.6%)
Total percent agreement: 95.9% (95% CI: 93.3 – 97.7%)

Equivocal results evaluated as negative:

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Equivocal results evaluated as positive:

		QuantaLite Scl-70 ELISA (Units)
		Positive >20	Negative <20	Total
EliA Scl-70S(EliA U/mL)	Positive >10	53	21	74
	Negative ≤10	4	296	300
	Total	57	317	374
Positive percent agreement:93.0% (95% CI: 82.2 – 97.8%)

Negative percent agreement: 93.4% (95% Cl: 89.9 - 95.8%) Total percent agreement: 93.3% (95% CI: 90.2 - 95.5%)

b. Matrix comparison:

Serum, lithium heparin plasma, and EDTA plasma were collected from the same patients (n = 50) to demonstrate that the plasma results do not deviate from the corresponding serum results and are within the pre-defined specifications.

Samples were spiked with a serum sample of high antibody titer to cover the measuring range. Samples were tested in duplicates. Passing & Bablok regression plots were generated using the first replicate only and by plotting the concentration observed from the control tube (serum) versus the concentration for each test collection tube. The corresponding slopes of regression and coefficient determination are summarized in the tables below:

	Range tested(EliA U/mL)	Slope(95% CI)	Intercept(95% CI)	R2
Serum vs heparinplasma	0.6 — 278.9	1.05(1.03 to 1.07)	-0.08(-0.24 to +0.17)	1.00
Serum vs EDTAplasma	0.6 — 264.2	1.00(0.99 to 1.02)	-0.05(-0.29 to +0.21)	1.00

C. Instrument comparison

Performance of EliA Scl-70° was evaluated on the Phadia 250 instruments using 24 positive, 8 equivocal and 4 negative samples were analyzed in six runs in single replicates on three Phadia 250 instruments, with 2 runs on each instrument. The regression analysis results are summarized as follows:

	Intercept	Slope
Estimate	-0.07	0.96
95% Cl	-0.52 – 0.14	0.94 – 0.99

3. Clinical studies:

Clinical Sensitivity and specificity: a.

336 clinically defined sera from patients with scleroderma or other connective tissue diseases, bacterial or viral infections, cancer or rheumatoid arthritis were tested on EliA Scl-70° as laid out in the respective Directions for Use

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Test results of EliA Scl-70 with 336 clinically defined samples. The diagnostic group of Scleroderma diagnosed patients (SSc) is the positive target group. Equivocal results evaluated as negative:

	Diagnostic Group - SSc
	+ / SSc	- / Controls*	total
Positive test >10 EliA U/mL	31	3	34
Negative test ≤10 EliA U/mL	70	232	302
Total	101	235	336

Sensitivity: 30.7% (95% CI: 22.1% - 40.8%) Specificity: 98.7% (95% CI: 96.0% - 99.7%)

The table below shows the positive results for each clinical subgroup. Equivocal results evaluated as negative:

Disease	Total numberof samples	Number & percentage of positive samplesQuantaLite Scl-70		Elia Scl-70s
SSc	101	29	29%	31	31%
CREST	33	2	6%	2	6%
MCTD	37	0	0%	0	0%
SLE	34	1	3%	0	0%
SS	26	1	4%	1	4%
PM/DM	5	0	0%	0	0%
RA	30	0	0%	0	0%
Cancer	20	0	0%	0	0%
Infections	50	2	4%	0	0%

SSc: Scleroderma, CREST: limited cutaneous form of systemic sclerosis, MCTD: Mixed connective tissue disease, SLE: Systemic Lupus Erythematosus, SS: Sjögren's syndrome- systemic sclerosis, PM/DM: Poly-/Dermatomyositis, RA: Rheumatoid arthritis, Cancer: various cancers, Infections: various bacterial and viral infections.

Other clinical supportive data (when a. is not applicable): b. Not applicable

1. Clinical cut-off:
  Same as assay cut-off
1. Expected values/Reference range:
  Antibody prevalence in autoimmune patients varies widely depending on disease area. The proportion of sera from a normal population found positive for Scl-70 antibodies covered by the EliA Scl-70 test is below 1 %. Expected values may vary depending on the population tested.

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).