EliA anti-TG Immunoassay is intended for the in vitro quantitative measurement of IgG antibodies directed to thyroglobulin (TG) in human serum and plasma (Li-heparin, EDTA) as an aid in the clinical diagnosis of autoimmune thyroiditis and Graves' disease in conjunction with other laboratory and clinical findings. EliA anti-TG uses the EliA IgG method on the instrument Phadia 250.

EliA anti-TPO Immunoassay is intended for the in vitro quantitative measurement of IgG antibodies directed to thyroid peroxidase (TPO) in human serum and plasma (Li-heparin, EDTA) as an aid in the clinical diagnosis of autoimmune thyroiditis and Graves' disease in conjunction with other laboratory and clinical findings. EliA anti-TPO uses the EliA IgG method on the instrument Phadia 250.

EliA Thyroid Positive Control 250 is intended for laboratory use in monitoring the performance of in vitro measurement of antibodies against thyroid peroxidase (TPO) and thyroglobulin (TG) with Phadia 250 using the EliA IgG method.

EliA Thyroid Positive Control 2500/5000 is intended for laboratory use in monitoring the performance of in vitro measurement of antibodies against thyroid peroxidase (TPO) and thyroglobulin (TG) with Phadia 2500/5000 using the EliA IgG method.

Device Description

The method specific reagents on Phadia® 250 and Phadia® 2500/5000 are identical: they are only filled in different containers. Each device consists of:

EliA anti-TG wells are coated with a human thyroglobulin antigen 4 carriers (16 wells each), ready to use; or EliA anti-TPO wells are coated with a human recombinant thyroid peroxidase
antigen 4 carriers (16 wells each), ready to use; EliA Sample Diluent: PBS containing BSA, detergent and 0.095% sodium azide - 6 bottles, 48 mL each, ready to use; or 6 bottles, 400 mL each, ready to use;
-EliA IgG Conjugate 50 or 200: ß-Galactosidase labeled anti-IgG (mouse monoclonal antibodies) in PBS containing BSA and 0.06% sodium azide - 6 wedge shaped bottles, 5 mL each, ready to use; or 6 wedge shaped bottles, 19 mL each, ready to use
-EliA Thyroid Positive Control 250 or 2500/5000: Human serum containing IqG antibodies to TG and TPO in PBS containing BSA, detergent and 0.095% sodium azide - 6 single use vials, 0.3 mL each, ready to use;
EliA Negative Control 250 or 2500/5000: Human sera from healthy donors in -PBS containing BSA, detergent and 0.095% sodium azide - 6 single-use vials, 0.3 mL each, ready to use;
-EliA IgG Calibrator Strips: Human IqG (0, 4, 10, 20, 100, 600 µq/L) in PBS containing BSA, detergent and 0.095% sodium azide - 5 strips, 6 single-use vials per strip, 0.3 mL each, ready to use;
-EliA IgG Curve Control Strips: Human IgG (20 µg/L) in PBS containing BSA, detergent and 0.095% sodium azide - 5 strips, 6 single-use vials per strip, 0.3 mL each, ready to use;
-EliA IqG Calibrator Well: Coated with mouse monoclonal antibodies - 4 carriers (12 wells each), ready to use.

The Phadia EliA Immunodiagnostic System is an 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 anti-TG or anti-TPO test.

AI/ML Overview

Here's an analysis of the acceptance criteria and the study proving the device meets them, based on the provided text.

1. Table of Acceptance Criteria and Reported Device Performance

The acceptance criteria are not explicitly stated as a formal table with pass/fail values. Instead, the document presents performance characteristics and demonstrates that the device meets these characteristics, implying they are the de facto acceptance criteria. I will infer these criteria from the studies conducted.

EliA anti-TG Immunoassay

Acceptance Criterion (Inferred)	Reported Device Performance (Phadia 250)	Reported Device Performance (Phadia 2500/5000)
Precision (Total Imprecision CV%)
Low Concentration (30.6 IU/mL)	11.9%	17.8%
Mid-range Concentration (184.8 IU/mL)	3.1%	5.6%
High Concentration (4147.2 IU/mL)	4.7%	10.2%
Linearity/Reportable Range	12 – 4794 IU/mL	12 – 4794 IU/mL
Hook Effect	No hook effect observed up to 27.7 times above upper limit	No hook effect observed up to 27.7 times above upper limit
Limit of Detection (LoD)	12.0 IU/mL (single shared LoD)	12.0 IU/mL (single shared LoD)
Interference (Bilirubin F/C, Hemoglobin, Lipemic factor, Rheumatoid factor, Thyroxine, Iodide)	No interference observed up to specified concentrations (ratios of blank/spiked 0.94 – 1.09)	No interference observed up to specified concentrations (ratios of blank/spiked 0.94 – 1.09)
Reference Sera Evaluation (CAP, NEQAS)	All targets hit ("OK")	All targets hit ("OK")
Carry-over	Negligible effect	None (disposable tips)
Method Comparison (vs. Predicate VarelisA TG)
Positive Percent Agreement (equivocal as negative)	89.7% (95% CI: 83.3% -94.3%)	Not separately reported, assumed similar
Negative Percent Agreement (equivocal as negative)	88.7% (95% CI: 84.6% -92.1%)	Not separately reported, assumed similar
Total Percent Agreement (equivocal as negative)	89.0% (95% CI: 85.7% -91.8%)	Not separately reported, assumed similar
Positive Percent Agreement (equivocal as positive)	86.3% (95% CI: 80.7% - 90.8%)	Not separately reported, assumed similar
Negative Percent Agreement (equivocal as positive)	91.3% (95% CI: 87.0% -94.5%)	Not separately reported, assumed similar
Total Percent Agreement (equivocal as positive)	89.0% (95% CI: 85.7% - 91.8%)	Not separately reported, assumed similar
Matrix Comparison (Serum vs. EDTA Plasma)	Slope: 1.00 (0.97 to 1.03), R2: 1.00	Not separately reported, assumed similar
Matrix Comparison (Serum vs. Li-heparin Plasma)	Slope: 1.00 (0.97 - 1.03), R2: 1.00	Not separately reported, assumed similar
Instrument Comparison (Phadia 250 vs. Phadia 2500/5000)	Slope: 0.96 (0.93 - 0.97), Intercept: 1.7 (0.7 - 3.8)	Not separately reported, assumed compared to Phadia 250
Clinical Sensitivity (AI Thyroiditis)	55.8% (95% CI: 48.9% - 62.6%)	Not separately reported, assumed similar
Clinical Specificity (AI Thyroiditis)	88.9% (95% CI: 85.8% - 91.5%)	Not separately reported, assumed similar

EliA anti-TPO Immunoassay

Acceptance Criterion (Inferred)	Reported Device Performance (Phadia 250)	Reported Device Performance (Phadia 2500/5000)
Precision (Total Imprecision CV%)
Low Concentration (15.7 IU/mL)	8.7%	13.1%
Mid-range Concentration (66.7 IU/mL)	4.5%	7.0%
High Concentration (1212.6 IU/mL)	6.2%	9.5%
Linearity/Reportable Range	4 – 1542 IU/mL	4 – 1542 IU/mL
Hook Effect	No hook effect observed up to 13.4 times above upper limit	No hook effect observed up to 13.4 times above upper limit
Limit of Detection (LoD)	4.0 IU/mL (single shared LoD)	4.0 IU/mL (single shared LoD)
Interference (Bilirubin F/C, Hemoglobin, Lipemic factor, Rheumatoid factor, Thyroxine, Iodide)	No interference observed up to specified concentrations (ratios of blank/spiked 0.93 – 1.05)	No interference observed up to specified concentrations (ratios of blank/spiked 0.93 – 1.05)
Reference Sera Evaluation (CAP, NEQAS)	All targets hit ("OK")	All targets hit ("OK")
Carry-over	Negligible effect	None (disposable tips)
Method Comparison (vs. Predicate VarelisA TPO)
Positive Percent Agreement (equivocal as negative)	100% (95% CI: 97.8% – 100.0%)	Not separately reported, assumed similar
Negative Percent Agreement (equivocal as negative)	89.7% (95% CI: 85.9% -92.8%)	Not separately reported, assumed similar
Total Percent Agreement (equivocal as negative)	93.2% (95% CI: 90.6% -95.2%)	Not separately reported, assumed similar
Positive Percent Agreement (equivocal as positive)	100% (95% CI: 98.3% – 100.0%)	Not separately reported, assumed similar
Negative Percent Agreement (equivocal as positive)	73.7% (95% CI: 68.2% -78.7%)	Not separately reported, assumed similar
Total Percent Agreement (equivocal as positive)	84.9% (95% CI: 81.5% -88.8%)	Not separately reported, assumed similar
Matrix Comparison (Serum vs. EDTA Plasma)	Slope: 1.00 (0.90 to 1.10), R2: 0.99	Not separately reported, assumed similar
Matrix Comparison (Serum vs. Li-heparin Plasma)	Slope: 0.99 (0.95 - 1.03), R2: 0.99	Not separately reported, assumed similar
Instrument Comparison (Phadia 250 vs. Phadia 2500/5000)	Slope: 0.98 (0.97 – 0.99), Intercept: 0.2 (-0.4 – 0.8)	Not separately reported, assumed compared to Phadia 250
Clinical Sensitivity (AI Thyroiditis)	82.3% (95% CI: 76.6% – 87.2%)	Not separately reported, assumed similar
Clinical Specificity (AI Thyroiditis)	90.5% (95% CI: 87.5% – 92.9%)	Not separately reported, assumed similar

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

Precision/Reproducibility:
- EliA anti-TG/TPO on Phadia 250/2500/5000: 8 samples, each with 252 replicate determinations (21 runs x 3 instruments x 7 runs each over 7 days).
- Data Provenance: Not explicitly stated, implied to be internal lab studies.
Linearity/Assay Reportable Range:
- EliA anti-TG/TPO on Phadia 250/2500/5000: 6-7 patient serum samples.
- Data Provenance: Not explicitly stated, implied to be internal lab studies.
Detection Limit (LoB/LoD):
- EliA anti-TG/TPO on Phadia 250/2500/5000: Four analyte-free samples and four low antibody concentration blood donor samples. Each measured in 36 replicates (6 replicates x 6 runs). Total of 8 samples x 36 replicates = 288 measurements.
- Data Provenance: Not explicitly stated, implied to be internal lab studies using blood donors.
Endogenous Interference:
- EliA anti-TG/TPO: Three serum samples (one negative, one near cut-off, one high positive). Each spiked with different interfering substances or blanks and analyzed in triplicates. Runs repeated twice.
- Data Provenance: Not explicitly stated, implied to be internal lab studies.
Reference Sera:
- EliA anti-TG: 8 samples from CAP.
- EliA anti-TPO: 8 samples from CAP and 12 samples from UK-NEQAS.
- Data Provenance: External proficiency testing programs (CAP, UK-NEQAS).
Carry-over:
- Phadia 250: A serum sample (diluted 1:2 and 1:20). Tested with EliA Ro (another assay) to demonstrate instrument's general carry-over performance.
- Data Provenance: Not explicitly stated, implied to be internal lab studies.
Assay Cut-off / Expected Values:
- EliA anti-TG/TPO: 604 apparently healthy blood donor samples.
- Data Provenance: Caucasian, African American, Hispanic, and Asian individuals, almost equally distributed by sex and age. (Source country not specified but "blood donor samples" are typically collected prospectively for such studies).
Method Comparison and Clinical Sensitivity/Specificity:
- EliA anti-TG/TPO: 718 serum samples from patients with various thyroid and autoimmune conditions (Graves' disease, autoimmune thyroiditis, non-AI thyroid disease, connective tissue disease, Crohn's disease, ulcerative colitis, primary biliary cirrhosis, HIV, HCV, HBV, other infection, cancer, rheumatoid arthritis, hypergamma-globulinemia, systemic lupus erythematosus, Sjögren's syndrome, celiac disease, type 1 diabetes mellitus, type II diabetes mellitus, pregnant women of all trimesters, pre-eclampsia, miscarriage, thyroid cancer, myasthenia gravis, pernicious anemia, chronic lymphocytic thyroiditis, sub-acute thyroiditis, multi-nodular goiter).
- Data Provenance: Not explicitly stated (e.g., country of origin, retrospective/prospective).
Matrix Comparison:
- EliA anti-TG/TPO: 57 patients from whom serum, lithium heparin plasma, and EDTA plasma were collected.
- Data Provenance: Not explicitly stated, implied to be patient samples.

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

The document does not specify the use of "experts" in the context of diagnostic performance studies. For immunoassay devices like these, ground truth is typically established by:

Reference Methods: Comparison to a legally marketed predicate device (VarelisA TG Antibodies, VarelisA TPO Antibodies) as seen in the method comparison study.
Clinical Diagnosis: For sensitivity and specificity, samples are categorized based on "diagnosis" (e.g., Graves' disease, autoimmune thyroiditis). This implies a clinical ground truth, but the details of how these diagnoses were established (e.g., by how many clinicians, their specialties, or experience levels) are not provided.
External Reference Sera: For reference sera evaluation, targets are established by external institutions like CAP and UK-NEQAS, which rely on peer groups or provider definitions.

Therefore, there's no mention of a specific number of experts or their qualifications establishing ground truth in the way one might see for image-based diagnostics.

4. Adjudication Method for the Test Set

The document does not describe an explicit adjudication method for establishing ground truth, such as 2+1 or 3+1 expert consensus. Instead, it relies on established clinical diagnoses for sensitivity/specificity studies and predicate device results for method comparison.

5. If a Multi-Reader Multi-Case (MRMC) Comparative Effectiveness Study was done, If so, what was the effect size of how much human readers improve with AI vs without AI assistance

No MRMC comparative effectiveness study was done. This device is an in vitro diagnostic (IVD) immunoassay, not an AI-assisted diagnostic tool for human readers. Its performance is evaluated intrinsically and in comparison to a predicate device, not in terms of human reader improvement.

6. If a Standalone (i.e. algorithm only without human-in-the-loop performance) was done

The device is a standalone immunoassay system (EliA anti-TG Immunoassay and EliA anti-TPO Immunoassay run on Phadia 250/2500/5000 instruments). Its performance is inherently "standalone" in the sense of being an automated laboratory test without direct human interpretive input at the point of result generation. Results are quantitative values that clinicians interpret. Therefore, the entire analytical and clinical performance section describes the device's standalone performance.

7. The Type of Ground Truth Used

Clinical Diagnosis: For clinical sensitivity and specificity studies, samples were linked to known diagnoses such as "Graves' Disease" and "Autoimmune Thyroiditis." The basis for these diagnoses (e.g., pathology, clinical outcomes, or expert opinion) is not detailed beyond being "clinically defined."
Predicate Device: For method comparison, the reference standard was the legally marketed predicate device (VarelisA TG Antibodies and VarelisA TPO Antibodies).
External Program Targets: For reference sera, the ground truth was the targets set by CAP (College of American Pathologists) and UK-NEQAS (United Kingdom National External Quality Assessment Service). These targets are often established by peer consensus or by the program providers.

8. The Sample Size for the Training Set

The document describes studies for validation of performance characteristics but does not explicitly mention a "training set" in the context of machine learning or AI development. Since these are immunoassays, they are based on biochemical reactions and calibration rather than a machine learning model that requires training data. The calibration process uses specific calibrator materials.

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

As this is an immunoassay and not an AI/ML device, the concept of a "training set" with established ground truth doesn't apply in the same way. The device is calibrated using:

EliA IgG Calibrator Strips: Human IgG at defined concentrations (0, 4, 10, 20, 100, 600 µq/L). These calibrators are traceable to the International Reference Preparation (IRP) 67/86 of Human Serum Immunoglobulins A, G and M from WHO. New batches are compared to a secondary standard or the IRP directly to ensure correct concentration. This traceability constitutes the "ground truth" for calibration.
EliA IgG Curve Control Strips: Human IgG (20 µg/L) for monitoring calibration curve performance.

The "ground truth" for the calibrators is therefore established through established international reference standards and their traceability.

Summary

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

March 25, 2016

Phadia US Inc. Martin Mann Sr. Regulatory Affairs Manager 4169 Commercial Avenue Portage, MI 49002

Re: K151799 Trade/Device Name: EliATM anti-TG Immunoassay EliA™ anti-TPO Immunoassay EliATM Thyroid Positive Control 250 EliATM Thyroid Positive Control 2500/5000 Regulation Number: 21 CFR §866.5870 Regulation Name: Thyroid autoantibody immunological test system Regulatory Class: II Product Code: JZO, JJY Dated: February 24, 2016 Received: February 25, 2016

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 Parts 801 and 809); medical device reporting (reporting of medical device-related adverse events) (21 CFR 803); good manufacturing practice requirements

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Page 2-Mr. Martin Mann

as set forth in the quality systems (OS) regulation (21 CFR Part 820); and if applicable, the electronic product radiation control provisions (Sections 531-542 of the Act); 21 CFR 1000-1050.

If you desire specific advice for your device on our labeling regulations (21 CFR Parts 801 and 809), 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/ResourcesforYou/Industry/default.htm. Also, please note the regulation entitled. "Misbranding by reference to premarket notification" (21 CFR Part 807.97). For questions regarding the reporting of adverse events under the MDR regulation (21 CFR Part 803), please go to

http://www.fda.gov/MedicalDevices/Safety/ReportaProblem/default.htm for 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 yours,

Leonthena R. Carrington -S

Leonthena R. Carrington, MS, MBA, MT(ASCP) 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) K151799

Device Name

EliA™ anti-TG Immunoasay, EliA™ anti-TPO Immunoassay, EliA™ Thyroid Positive Control 250, and EliA™ Thyroid Positive Control 2500/5000.

Indications for Use (Describe)

Type of Use (Select one or both, as applicable)

X Prescription Use (Part 21 CFR 801 Subpart D)

Over-The-Counter Use (21 CFR 801 Subpart C)

CONTINUE ON A SEPARATE PAGE IF NEEDED.

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F.1 510k Decision Summary Input

510(k) SUBSTANTIAL EQUIVALENCE DETERMINATION DECISION SUMMARY

A. 510(k) Number: K151799

B. Purpose for Submission: New devices
C. Measurand: lgG Antibodies to TG and TPO

D. Type of Test: Quantitative immunofluorescence assays

E. Applicant: Phadia AB, Sweden

F. Proprietary and Established Names:

EliA™ anti-TG Immunoassay EliA™ anti-TPO Immunoassay EliA™ Thyroid Positive Control 250 EliA™ Thyroid Positive Control 2500/5000

G. Requlatory Information:

1. Regulation section:
  21 CFR §866.5870, Thyroid autoantibody immunological test system 21 CFR §862.1660, Quality control material (assayed and unassayed)
Classification: 2.
Class II (Assays) Class I (Controls)
1. Product code:
  JZO System, Test, Thyroid Autoantibody JJY Multi-Analyte Controls, All Kinds (assayed)
1. Panel:
  Immunology

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H. Intended Use:

Intended use(s):

EliA anti-TG is intended for the in vitro quantitative measurement of IgG antibodies directed to thyroglobulin (TG) in human serum and plasma (Li-heparin, EDTA) as an aid in the clinical diagnosis of autoimmune thyroiditis and Graves' disease in conjunction with other laboratory and clinical findings. EliA anti-TG uses the EliA IgG method on the instruments Phadia 250 and Phadia 2500/5000.

EliA anti-TPO is intended for the in vitro quantitative measurement of IgG antibodies directed to thyroid peroxidase (TPO) in human serum and plasma (Li-heparin, EDTA) as an aid in the clinical diagnosis of autoimmune thyroiditis and Graves' disease in conjunction with other laboratory and clinical findings. EliA anti-TPO uses the EliA IgG method on the instruments Phadia 250 and Phadia 2500/5000.

EliA Thyroid Positive Control 250 and EliA Thyroid Positive Control 2500/5000 is intended for laboratory use in monitoring the performance of in vitro measurement of antibodies against thyroid peroxidase (TPO) and thyroglobulin (TG) with Phadia 250 and Phadia 2500/5000 using the EliA IgG method.

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

Special conditions for use statement(s): For prescription use only

4.__Special instrument requirements:
Phadia® 250 or Phadia® 2500/5000

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-Device Description:

The method specific reagents on Phadia® 250 and Phadia® 2500/5000 are identical: they are only filled in different containers. Each device consists of:

EliA anti-TG wells are coated with a human thyroglobulin antigen 4 carriers (16 wells each), ready to use; or EliA anti-TPO wells are coated with a human recombinant thyroid peroxidase
antigen 4 carriers (16 wells each), ready to use; EliA Sample Diluent: PBS containing BSA, detergent and 0.095% sodium azide - 6 bottles, 48 mL each, ready to use; or 6 bottles, 400 mL each, ready to use;
-EliA IgG Conjugate 50 or 200: ß-Galactosidase labeled anti-IgG (mouse monoclonal antibodies) in PBS containing BSA and 0.06% sodium azide - 6 wedge shaped bottles, 5 mL each, ready to use; or 6 wedge shaped bottles, 19 mL each, ready to use
-EliA Thyroid Positive Control 250 or 2500/5000: Human serum containing IqG antibodies to TG and TPO in PBS containing BSA, detergent and 0.095% sodium azide - 6 single use vials, 0.3 mL each, ready to use;
EliA Negative Control 250 or 2500/5000: Human sera from healthy donors in -PBS containing BSA, detergent and 0.095% sodium azide - 6 single-use vials, 0.3 mL each, ready to use;
-EliA IgG Calibrator Strips: Human IqG (0, 4, 10, 20, 100, 600 µq/L) in PBS containing BSA, detergent and 0.095% sodium azide - 5 strips, 6 single-use vials per strip, 0.3 mL each, ready to use;
-EliA IgG Curve Control Strips: Human IgG (20 µg/L) in PBS containing BSA, detergent and 0.095% sodium azide - 5 strips, 6 single-use vials per strip, 0.3 mL each, ready to use;
-EliA IqG Calibrator Well: Coated with mouse monoclonal antibodies - 4 carriers (12 wells each), ready to use.

J. Substantial Equivalence Information:

1. Predicate device name(s) and 510(k) number(s):
VarelisA TG Antibodies, Phadia GmbH	K003414
VarelisA TPO Antibodies, Phadia GmbH	K993585
EliATM IgG/IgM/IgA Negative Control 250	K091845
EliA IgG Calibrator Strips	K061165

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2. Comparison with predicate device:

EliA anti-TG Immunoassay – Similarities to predicate device

Feature	Predicate DeviceVarelisA TG	New DeviceEliA anti-TG
Intended Use	The VarelisA TG (Thyroglobulin)Antibodies EIA kit is designed forthe quantitative and qualitativedetermination of thyroglobulinantibodies in serum or plasma toaid in the diagnosis of thyroiddiseases such as autoimmunethyroiditis and Graves' disease	EliA anti-TG is intended for the invitro quantitative measurementof IgG antibodies directed tothyroglobulin (TG) in humanserum and plasma (Li-heparin,EDTA) as an aid in the clinicaldiagnosis of autoimmunethyroiditis and Graves' disease inconjunction with other laboratoryand clinical findings. EliA anti-TGuses the EliA IgG method on theinstruments Phadia 250 andPhadia 2500/5000.
Internal Controls	Positive and Negative Controlincluded in the kit	Positive and negative Controlprovided with the EliA ThyroidPositive Control 250 / 2500/5000and EliA IgG/IgM/IgA NegativeControl 250 / 2500/5000,respectively
Assay Type	ELISA
Type of test	quantitative
Reported Unit	IU/ml
Solid Phase	Microwells
Antigen	human thyroglobulin antigen
Feature	Predicate DeviceVarelisA TG	New DeviceEliA anti-TG
Instrumentation	ELISA-Reader needed	Phadia 250 and 2500/5000 arefully automated immunoassayanalyzers
Reactiontemperature	Room temperature	37°C controlled
Incubation times	Positive and negative Controls,diluted patient samples: 30 min.Conjugate: 30 min.Substrate: 10 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 IgGß-Galactosidase (mousemonoclonal antibodies)
Signal	Optical density (at 450nm)	Fluorescence
Calibration	6-point Calibration	6-point total IgG Calibration
Calibrators	6 vials of TG-specific IgG atconcentrations of0 - 30 - 100 - 300 - 1000 -3000 IU/ml	6 vials of human IgG atconcentrations 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 andgeneral reagents)
Sample Dilution	1:101	1:100
Cut-off	< 60 IU/ml negative60-100 IU/ml equivocal> 100 IU/ml positive	< 40 IU/ml negative40-60 IU/ml equivocal> 60 IU/ml positive
Substrate	TMB Chromogen	Development Solution 0.01 %4-Methylumbelliferyl-β-D-galactoside & <0.0010%preservative (mixture of 5-chloro-2-methyl-2H-isothiazol-3-one[EC no. 247-500-7] and 2-methyl-2H-isothiazol-3-one [ECno. 220-239-6] (3:1))
Feature	Predicate Device	New Device
Intended Use	VarelisA TPOThe VarelisA TPO AntibodiesEIA kit is designed for thequantitative and qualitativedetermination of TPO (thyroidperoxidase) antibodies in serumor plasma to aid in the diagnosisof thyroid diseases such asautoimmune thyroiditis andGraves' disease.	EliA anti-TPOEliA anti-TPO is intended for thein vitro quantitativemeasurement of IgG antibodiesdirected to thyroid peroxidase(TPO) in human serum andplasma (Li-heparin, EDTA) as anaid in the clinical diagnosis ofautoimmune thyroiditis andGraves' disease in conjunctionwith other laboratory and clinicalfindings. EliA anti-TPO uses theEliA IgG method on theinstruments Phadia 250 andPhadia 2500/5000.
Internal Controls	Positive and Negative Controlincluded in the kit	Positive and negative Controlprovided with the EliA ThyroidPositive Control 250 / 2500/5000and EliA IgG/IgM/IgA NegativeControl 250 / 2500/5000,respectively
Assay Type	ELISA
Type of test	quantitative
Reported Unit	IU/ml
Solid Phase	Microwells
Antigen	human recombinant TPO antigen
Feature	Predicate DeviceVarelisA TPO	New DeviceEliA anti-TPO
Instrumentation	ELISA-Reader needed	Phadia 250 and 2500/5000 arefully automated immunoassayanalyzers
Reactiontemperature	Room temperature	37°C controlled
Incubation times	Positive and negative Controls,diluted patient samples: 30 min.Conjugate: 30 min.Substrate: 10 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 IgGß-Galactosidase (mousemonoclonal antibodies)
Signal	Optical density (at 450nm)	Fluorescence
Calibration	6-point antigen-specificCalibration	6-point total IgG Calibration
Calibrators	6 vials of TPO-specific IgG atconcentrations of0 - 30 - 100 - 300 - 1000 -3000 IU/ml	6 vials of human IgG atconcentrations 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 andgeneral reagents)
Sample Dilution	1:101	1:200
Cut-off	< 60 IU/ml negative60-100 IU/ml equivocal> 100 IU/ml positive	< 25 IU/ml negative25-35 IU/ml equivocal> 35 IU/ml positive
Substrate	TMB Chromogen	Development Solution 0.01 %4-Methylumbelliferyl-ß-D-galactoside & <0.0010%preservative (mixture of 5-chloro-2-methyl-2H-isothiazol-3-one[EC no. 247-500-7] and 2-methyl-2H-isothiazol-3-one [ECno. 220-239-61 (3:1))

{9}------------------------------------------------

EliA anti-TG – Differences to predicate device

{10}------------------------------------------------

EliA anti-TPO Immunoassay – Similarities to predicate device

{11}------------------------------------------------

EliA anti-TPO – Differences to predicate device

{12}------------------------------------------------

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™ test wells are coated with a human thyroglobulin protein, or a human recombinant thyroid peroxidase protein. If present in the patient's specimen, antibodies to TG or TPO bind to their specific antigen. After washing away nonbound 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 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:

a. Precision/Reproducibility:

To determine the precision of the assays on Phadia 250 and Phadia 2500/5000 instrument, the variability was assessed on 8 samples.

Two batches were used to determine the precision of the assays on Phadia 250 and on Phadia 2500/5000. The results are summarized in the tables below:

{13}------------------------------------------------

EliA anti-TG Immunoassay:

To determine the precision of the assay on the Phadia 250 instrument, 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 value	Intra Run	Inter Run	Total Imprecision
IU/mL	CV%	CV%	CV%
30.6	8.7	4.9	11.9
37.3	6.2	4.7	11.4
74.9	4.3	0.9	6.7
124.9	3.6	0.7	4.6
184.8	2.8	0.6	3.1
771.5	2.7	1.1	3.2
2065.1	3.3	1.4	4.3
4147.2	4.0	1.3	4.7

EliA anti-TG on Phadia 250 (n = 252)

To determine the precision of the assay on the Phadia 2500/5000 instrument, 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 valueIU/mL	Intra-RunCV%	Inter-RunCV%	Total ImprecisionCV%
31.4	13.5	3.2	17.8
38.9	11.9	2.3	16.0
76.7	5.2	1.9	9.2
134.5	5.3	2.0	8.3
193.0	4.3	2.0	5.6
754.2	5.2	1.2	5.4
1869.6	5.4	0.0	6.9
3797.5	8.9	1.3	10.2

EliA anti-TG on Phadia 2500/5000 (n = 252)

EliA anti-TPO Immunoassay:

{14}------------------------------------------------

Mean valueIU/mL	Intra RunCV%	Inter RunCV%	Total ImprecisionCV%
15.7	5.7	2.3	8.7
27.4	4.6	1.5	5.7
39.4	3.8	0.5	6.2
66.7	3.9	1.0	4.5
218.0	4.6	1.3	4.9
480.1	4.7	1.0	5.2
814.9	3.9	0.4	4.6
1212.6	4.0	0.0	6.2

EliA anti-TPO on Phadia 250 (n = 252)

Mean value	Intra-Run	Inter-Run	Total Imprecision
IU/mL	CV%	CV%	CV%
17.7	8.7	2.2	13.1
26.6	6.4	3.1	8.0
38.5	5.9	3.4	7.3
65.3	6.1	2.6	7.0
202.9	6.9	2.7	7.6
466.6	7.2	2.2	7.8
802.8	7.4	1.7	7.8
1340.2	9.0	2.1	9.5

EliA anti-TPO on Phadia 2500/5000 (n = 252)

{15}------------------------------------------------

b. Linearity/assay reportable range:

6-7 patient serum samples were diluted in sample diluent and tested with one batch of EliA anti-TG Immunoassay and EliA anti-TPO Immunoassay and one set of system reagents on Phadia 250 or Phadia 2500/5000. The ratios of observed/expected values were calculated. Depending on the lot specific EliA well factor, the upper limit of the measuring range may vary between different solid phase batches. The results are summarized below:

EliA anti-TG on Phadia 250

Dilution range(IU/mL)	Slope	Intercept	R2
2655.8 - 31.1	1.002	-1.46	0.9990
2935.0 - 33.5	1.008	13.21	0.9983
4827.9 - 62.5	1.025	58.32	0.9967
375.3 - 10.1*	0.998	1.01	0.9999
375.3 - 15.4	0.998	0.93	0.9999
468.8 - 17.5	0.995	1.34	0.9995
394.2 - 14.2	1.005	-0.07	0.9998
326.9 - 12.5	1.001	1.57	0.9994

*Evaluation including 1st dilution step below LoD

The claimed linear range for anti-TG is 12 – 4794 IU/mL

Dilution range(IU/mL)	Slope	Intercept	R2
2626.4 - 31.1	0.995	23.48	0.9993
2052.8 - 36.2	1.008	17.61	0.9991
5127.8 - 63.2	1.006	60.01	0.9981
375.9 - 11.1*	0.977	8.21	0.9963
375.9 - 17.6	0.971	9.70	0.9963
470.7 - 12.1	1.011	2.09	0.9992
412.0 - 15.4	0.987	-1.38	0.9990
327.9 - 13.6	1.016	4.09	0.9973

EliA anti-TG on Phadia 2500/5000

*Evaluation including 1st dilution step below LoD

The claimed linear range for anti-TG is 12 – 4794 IU/mL

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Dilution range(IU/mL)	Slope	Intercept	R2
1536.1 - 16.5	1.004	-1.78	0.9999
2201.2 - 22.1	1.048	-1.48	0.9946
479.8 - 7.0	1.008	-0.50	0.9999
310.9 - 3.2*	1.005	0.42	0.9998
310.9 - 4.9	1.005	0.55	0.9998
349.2 - 6.3	0.993	0.90	0.9998
458.0 - 5.6	0.998	1.33	0.9998

EliA anti-TPO on Phadia 250

*Evaluation including 1st dilution step below LoD

The claimed linear range for anti-TPO is 4 – 1542 IU/mL

Dilution range(IU/mL)	Slope	Intercept	R2
1400.8 - 15.1	0.987	-0.70	0.9993
1955.9 - 22.1	1.008	2.43	0.9997
471.6 - 4.6	0.988	1.55	0.9993
205.7 - 3.6*	1.071	0.40	0.9996
205.7 - 5.8	1.015	2.37	0.9979
349.4 - 5.8	0.997	0.90	0.9999
464.3 - 5.3	0.987	1.61	0.9994

EliA anti-TPO on Phadia 2500/5000

*Evaluation including 1st dilution step below LoD

The claimed linear range for anti-TPO is 4 - 1542 IU/mL

Hook Effect/Over the Range Results:

Hook effect was investigated by using five serum samples above the upper limit of the measuring range. Five high positive samples were diluted and the dilutions were measured in two replicates and compared to the upper limit of the technical measuring range (4794 IU/ml for EliA anti-TG and 1542 IU/ml for TPO EliA anti-TPO).

EliA anti-TG Immunoassay:

No hook effect was observed when analyzing five high positive samples that had concentrations up to 27.7 times above the upper limit of the technical measuring range.

EliA anti-TPO Immunoassay:

No hook effect was observed when analyzing five high positive samples that had concentrations up to 13.4 times above the upper limit of the technical measuring range.

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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.

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

The IgG calibrators are traceable (via unbroken chain of calibrations) to the International Reference Preparation (IRP) 67/86 of Human Serum Immunoqlobulins 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 uq/L. By using a conversion factor qiven by the lot-specific code of the EliA™ test well, the results are automatically converted to IU/mL for EliA anti-TG and EliA anti-TPO.

Stability:

The EliA IqG system reagents and the EliA IgG/IqM/IgA Negative Control are already FDA cleared under K063775 and K072393, respectively.

Shelf life:

An accelerated study was done to determine the shelf life for EliA anti-TG wells and EliA anti-TPO wells and it was determined to be 18 months. The real time stability test confirmed the determined shelf life.

On-board stability:

The on-board stability EliA anti-TG and EliA anti-TPO carriers (containing the antigen coated wells) was tested over 4 weeks using 3 positive and 2 negative samples only on the Phadia 250 instrument. As the storage conditions in the Phadia 2500/5000 instrument are similar the results can also be used for stability claims for the Phadia 2500/5000. The on-board stability for the Phadia 250 instrument was determined to be 28 days at 2-8°C.

Open Stability:

Stability of the foilbag containing the EliA anti-TG and EliA anti-TPO wells after first opening was tested and determined to be 9 months at 2-8°C.

{18}------------------------------------------------

d. Detection limit:

The limit of blank (LoB) and limit of detection (LoD) studies were done on both Phadia 250 and Phadia 2500/5000. Four different analyte-free samples and four blood donor samples with low antibody concentration were measured in six replicates in each of six runs at six different days (6 replicates x 6 runs = 36 replicates per sample). For both instrument types (Phadia 250 and Phadia 2500/5000), each sample was run in a total of six runs on two instruments (3 runs on instrument 1 and 3 runs on instrument 2) with two lots of wells and reagents. The results are summarized in the tables below:

EliA anti-TG (IU/mL)	LOB	LOD
Phadia 250	5.0	10.1
Phadia 2500/5000	4.9	8.5

It was decided to use a single LoD of 12.0 IU/mL for both instruments.

EliA anti-TPO (IU/mL)	LoB	LoD
Phadia 250	1.7	2.9
Phadia 2500/5000	1.5	2.8

It was decided to use a single LoD of 4.0 IU/mL for both instruments.

{19}------------------------------------------------

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

One neqative sample, one sample with concentration around the cut-off and one high positive sample were tested. The ratio of blank/spiked for equivocal and positive samples was 0.94 – 1.09 for EliA anti-TG and 0.93 – 1.05 for EliA anti-TPO. No interference was observed up to the concentrations listed in the table below:

Potential InterferingCompound	Concentration inundiluted sample
Bilirubin F	192 mg/dl
Bilirubin C	201 mg/dl
Hemoglobin	4960 mg/dl
Lipemic factor	1%
Rheumatoid factor	500 IU/ml
Thyroxine	2.5 ng/dl
lodide	3 mM

EliA anti-TG Immunoassay:

EliA anti-TPO Immunoassay:

Potential InterferingCompound	Concentration inundiluted sample
Bilirubin F	192 mg/dl
Bilirubin C	201 mg/dl
Hemoglobin	4960 mg/dl
Lipemic factor	1%
Rheumatoid factor	500 IU/ml
Thyroxine	2.5 ng/dl
Iodide	3 mM

{20}------------------------------------------------

Reference sera: Externally defined sera should be measured according to their target values as mentioned by the institution. For CAP, the target is set based on the complete peer group, if >80% consensus of all participants could be reached. For UK-NEQAS the target is set by the provider based on all responses; an equivocal target means that positive and negative responses are acceptable. The targets of the reference samples were hit.

Sample No.	Sample IDsupplier	Institution	Target	EliA anti-TG (IU/ml)
				Neg <40Equ 40-60Pos >60
1	S2-03	CAP	Pos	4511.7	OK
2	S2-11	CAP	Pos	5979.9	OK
3	S2-19	CAP	Neg	0.7	OK
4	S2-11	CAP	Pos	4650.7	OK
5	S2-19	CAP	Pos	6083.5	OK
6	S2-02	CAP	Pos	5333.6	OK
7	S2-10	CAP	Neg	0.8	OK
8	S2-18	CAP	Pos	3612.1	OK

Evaluation of QAS sera on EliA anti-TG

{21}------------------------------------------------

Sample No.	Sample ID supplier	Institution	Target	EliA anti-TPO (IU/ml)
				Neg <25Equ 25-35Pos >35
1	S2-03	CAP	Pos	150.1	OK
2	S2-11	CAP	Pos	171.3	OK
3	S2-19	CAP	Neg	0.0	OK
4	S2-11	CAP	Pos	97.0	OK
5	S2-19	CAP	Pos	164.4	OK
6	S2-02	CAP	Pos	155.6	OK
7	S2-10	CAP	Neg	0.0	OK
8	S2-18	CAP	Pos	89.6	OK
9	1012	NEQAS	Equ	41.4	OK
10	1032	NEQAS	Pos	102.1	OK
11	1042	NEQAS	Pos	136.5	OK
12	1052	NEQAS	Pos	98.3	OK
13	1062	NEQAS	Pos	87.5	OK
14	111-2	NEQAS	Pos	132.3	OK
15	112-2	NEQAS	Equ	21.8	OK
16	114-2	NEQAS	Pos	174.5	OK
17	116-2	NEQAS	Pos	90.4	OK
18	122-2	NEQAS	Pos	84.9	OK
19	123-2	NEQAS	Equ	1.6	OK
20	124-2	NEQAS	Pos	107.6	OK

Evaluation of QAS sera on EliA anti-TPO

{22}------------------------------------------------

Carry-over: 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 U/mL. The observed carry over effect is therefore negligible without any influence to assay results.

Phadia 2500/5000 instruments use disposable tips for pipetting samples and a separate pipette for the conjugate, therefore carry-over from samples to conjugate is impossible.

f. Assay cut-off:
A study was done on 604 apparently healthy blood donor samples from Caucasian, African American, Hispanic and Asian individuals almost equally distributed by sex and age in order to evaluate expected values for each of the antigens in the submission in the normal population and to confirm the defined cut-off. The samples were measured on the Phadia 250 instrument for EliA anti-TG and EliA anti-TPO. The 90t" and 95th percentile of the 604 samples were calculated and the 90th percentile was taken into account for setting the cut-off. The following values were selected for the cut-off:

EliA anti-TG
<40 IU/mL	Negative
40 – 60 IU/mL	Equivocal
>60 IU/mL	Positive

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

EliA anti-TPO
<25 IU/mL	Negative
25 — 35 IU/mL	Equivocal
>35 IU/mL	Positive

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

1. Comparison studies:
Method comparison with predicate device: a.

EliA anti-TG Immunoassay:

{23}------------------------------------------------

systemic lupus erythematosus (n = 24), Sjögren's syndrome (n = 14), celiac disease (n = 15), type 1 diabetes mellitus (n = 15), type II diabetes mellitus (n = 15), pregnant women of all trimesters (n = 90), pre-eclampsia (n = 5), miscarriage (n=9), thyroid cancer (n = 21), myasthenia gravis (n = 7), pernicious anemia (n = 8), chronic lymphocytic thyroiditis (n = 10), sub-acute thyroiditis (n = 5), multi-nodular goiter (n = 4).

Samples were analyzed with the EliA anti-TG and VarelisA TG assays. The test was run in single determination and values outside the overlapping measuring range for both tests (12-3000 IU/ml) were excluded from statistical analyses (n=280). The results are summarized in the tables below:

		VarelisA TG (IU/mL)
		Positive >100	Negative <100	Total
EliA anti-TG(IU/mL)	Positive >60	122	34	156
	Negative ≤60	14	268	282
	Total	136	302	438

EliA anti-TG - Equivocal results evaluated as negative:

Positive percent agreement: 89.7% (122/136) (95% Cl: 83.3% -94.3%)

Negative percent agreement: 88.7% (268/302) (95% CI: 84.6% -92.1%)

Total percent agreement: 89.0% [(122+268)/438] (95% Cl: 85.7% -91.8%)

EliA anti-TG - Equivocal results evaluated as positive:

		VarelisA TG (IU/mL)
		Positive >60	Negative <60	Total
EliA anti-TG	Positive ≥40	170	21	191
(IU/mL)	Negative <40	27	220	247
	Total	197	241	438

Positive percent agreement: 86.3% (170/197) (95% CI: 80.7% - 90.8%) Negative percent agreement: 91.3% (220/241) (95% CI: 87.0% -94.5%)

Total percent agreement: 89.0% [(170+220)/438] (95% Cl: 85.7% – 91.8%)

{24}------------------------------------------------

EliA anti-TPO Immunoassay:

A total of 718 serum samples were collected with a diagnosis from patients with Graves' disease (n = 100), autoimmune thyroiditis (n = 100), non-Al thyroid disease (n = 48), connective tissue disease (n = 15), Crohn`s disease (n = 20), ulcerative colitis (n = 20), primary biliary cirrhosis (n = 10), HIV infection (n = 20), HCV infection (n = 20), HBV infection (n = 20), other infection (n = 20), cancer (n = 56), rheumatoid arthritis (n = 12), hypergamma-globulinemia (n = 15), systemic lupus erythematosus (n = 24), Sjögren's syndrome (n = 14), celiac disease (n = 15), type 1 diabetes mellitus (n = 15), type II diabetes mellitus (n = 15), pregnant women of all trimesters (n = 90), pre-eclampsia (n = 5), miscarriage (n=9), thyroid cancer (n = 21), myasthenia gravis (n = 7), pernicious anemia (n = 8), chronic thyroiditis (n = 10), sub-acute thyroiditis (n = 5), multinodular goiter (n = 4).

Samples were analyzed with the EliA anti-TPO and VarelisA TPO assays. The test was run in single determination and values outside the overlapping measuring range for both tests (4-1542 IU/ml) were excluded from statistical analyses (n=297). The results are summarized in the tables below:

		VarelisA TPO (IU/mL)
		Positive >100	Negative <100	Total
EliA anti-	Positive ≥35	167	34	201
TPO (IU/mL)	Negative <35	0	297	297
	Total	167	331	498

EliA anti-TPO - Equivocal results evaluated as negative:

Positive percent agreement: 100% (167/167) (95% Cl: 97.8% –

100.0%)

Negative percent agreement: 89.7% (297/331) (95% CI: 85.9% -92.8%)

Total percent agreement: 93.2% [(167+297)/498] (95% Cl: 90.6% -95.2%)

EliA anti-TPO - Equivocal results evaluated as positive:
----------------------------------------------------------	--

		VarelisA TPO (IU/mL)
		Positive >100	Negative <100	Total
EliA anti-	Positive ≥25	213	75	288
TPO (IU/mL)	Negative <25	0	210	210
	Total	213	285	498

Positive percent agreement: 100% (213/213) (95% Cl: 98.3% – 100.0%) Negative percent agreement: 73.7% (210/285) (95% CI: 68.2% -78.7%)

Total percent agreement: 84.9% [(213+210)/498] (95% Cl: 81.5% -88.8%)

{25}------------------------------------------------

b. Matrix comparison:

Serum, lithium heparin plasma and EDTA plasma were collected from the same patients (n = 57) to demonstrate that the plasma results do not deviate from the corresponding serum results and are within the pre-defined specifications. Most samples were spiked with serum samples of high antibody titer to cover the measuring range; samples within the equivocal range were neat samples. Samples were tested in duplicates. Weighted Deming regression analysis was performed 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:

	EliA anti-TG Immunoassay:
--	---------------------------

	Range tested(IU/mL)	Slope(95% CI)	Intercept(95% CI)	R2
Serum vsEDTA	25.5 - 4323.7	1.00(0.97 to 1.03)	-3.16(-6.79 to -0.47)	1.00
Serum vsLi-heparin	24.3 - 4202.4	1.00(0.97 - 1.03)	-0.43(-3.96 - 3.11)	1.00

EliA anti-TPO Immunoassay:

	Range tested(IU/mL)	Slope(95% CI)	Intercept(95% CI)	R2
Serum vsEDTA	10.8 — 1445.2	1.00(0.90 to 1.10)	-3.07(-8.90 to 2.75)	0.99
Serum vsLi-heparin	11.0 — 1389.9	0.99(0.95 - 1.03)	-0.78(-2.25 to -0.68)	0.99

{26}------------------------------------------------

c. Instrument comparison

Performance of EliA™ anti-TG and EliA anti-TPO was evaluated on the Phadia 250 and Phadia 2500/5000 instruments using 30 positive, 8 equivocal and 12 negative samples. The samples were analyzed in six runs in single replicates on three Phadia 250 and three Phadia 2500/5000 instruments (2 runs on each instrument). The regression analysis results are summarized as follows:

EliA anti-TG Immunoassay:

	Intercept	Slope
Estimate	1 7	0.96
95% Cl	0.7 - 3.8	0.93 - 0.97

EliA anti-TPO Immunoassay:

	Intercept	Slope
Estimate	0.2	0.98
95% Cl	-0.4 – 0.8	0.97 – 0.99

1. Clinical studies:
a. Clinical sensitivity and specificity:

EliA anti-TG Immunoassay:

The clinically defined AI Thyroiditis samples that were used for the method comparison were also used to determine sensitivity and specificity of the assay. The results are summarized in the tables below.

EliA anti-TG - diagnostic group Al Thyroiditis - equivocal results evaluated as negative:

	Diagnostic Group - Al Thyroiditis
	+	-	total
Positive test >60.0 IU/mL	120	56	176
Negative test ≤60.0 IU/mL	95	447	542
Total	215	503	718

Sensitivity (95% CI): 55.8% (48.9% - 62.6%) Specificity (95% CI): 88.9% (85.8% - 91.5%)

{27}------------------------------------------------

The table below shows the results for each clinical subgroup:

Condition	Numberofsamples	No (%)pos. onEliA anti-TG	No (%)pos. onPredicateVarelisATG
Graves' Disease	100	48 (48%)	48 (48%)
Autoimmune thyroiditis	115	72 (62.6%)	69 (60%)
Non-AI Thyroid Disease	48	7 (14.6%)	9 (18.8%)
Connective Tissue Disease	15	2 (13.3%)	2 (13.3%)
Crohn's disease	20	1 (5%)	0 (0%)
Ulcerative colitis	20	1 (5%)	0 (0%)
Primary Biliary Cirrhosis	10	0 (0%)	0 (0%)
HIV infection	20	0 (0%)	3 (15%)
HCV infection	20	1 (5%)	2 (10%)
HBV infection	20	1 (5%)	2 (10%)
Other infection	20	1 (5%)	1 (5%)
Cancer	56	13 (23.2%)	7 (12.5%)
Rheumatoid arthritis	12	2 (16.7%)	1 (8.3%)
Hypergammaglobulinemia	15	1 (6.7%)	1 (6.7%)
Systemic lupus erythematosus	24	5 (20.8%)	3 (12.5%)
Sjögren's syndrome	14	2 (14.3%)	2 (14.3%)
Celiac disease	15	3 (20%)	2 (13.3%)
Type I diabetes mellitus	15	2 (13.3%)	2 (13.3%)
Type II diabetes mellitus	15	1 (6.7%)	1 (6.7%)
Pregnant women (all trimesters)	90	5 (5.6%)	2 (2.2%)
Pre-eclampsia	5	0 (0%)	0 (0%)
Miscarriage	9	6 (66.7%)	1 (11.1%)
Thyroid cancer	21	0 (0%)	0 (0%)
Myasthenia gravis	7	0 (0%)	0 (0%)
Pernicious anemia	8	2 (25%)	2 (25%)
Multi-nodular goiter	4	0 (0%)	0 (0%)

{28}------------------------------------------------

EliA anti-TPO Immunoassay:

EliA anti-TPO - diagnostic group Al Thyroiditis - equivocal results evaluated as negative:

Diagnostic Group - Al Thyroiditis
	+	-	total
Positive test >35.0 IU/mL	177	48	225
Negative test ≤35.0 IU/mL	38	455	493
Total	215	503	718

Sensitivity (95% CI): 82.3% (76.6% – 87.2%) Specificity (95% CI): 90.5% (87.5% – 92.9%)

{29}------------------------------------------------

The table below shows the results for each clinical subgroup:

Condition	Numberofsamples	No (%)pos. onEliA anti-TPO	No (%)pos. onPredicateVarelisATPO
Graves' Disease	100	81 (81%)	68 (68%)
Autoimmune thyroiditis	115	96 (83.5%)	88 (76.5%)
Non-AI Thyroid Disease	48	5 (10.4%)	5 (10.4%)
Connective Tissue Disease	15	1 (6.7%)	1 (6.7%)
Crohn's disease	20	0 (0%)	0 (0%)
Ulcerative colitis	20	0 (0%)	0 (0%)
Primary Biliary Cirrhosis	10	0 (0%)	0 (0%)
HIV infection	20	1 (5%)	0 (0%)
HCV infection	20	1 (5%)	1 (5%)
HBV infection	20	0 (0%)	0 (0%)
Other infection	20	0 (0%)	0 (0%)
Cancer	56	11 (19.6%)	5 (8.9%)
Rheumatoid arthritis	12	1 (8.3%)	1 (8.3%)
Hypergammaglobulinemia	15	0 (0%)	0 (0%)
Systemic lupus erythematosus	24	1 (4.2%)	0 (0%)
Sjögren's syndrome	14	1 (7.1%)	1 (7.1%)
Celiac disease	15	2 (13.3%)	2 (13.3%)
Type I diabetes mellitus	15	2 (13.3%)	2 (13.3%)
Type II diabetes mellitus	15	2 (13.3%)	2 (13.3%)
Pregnant women (all trimesters)	90	10 (11.1%)	10 (11.1%)
Pre-eclampsia	5	4 (80%)	3 (60%)
Miscarriage	9	2 (22.2%)	1 (11.1%)
Thyroid cancer	21	2 (9.5%)	1 (4.8%)
Myasthenia gravis	7	1 (14.3%)	0 (0%)
Pernicious anemia	8	1 (12.5%)	0 (0%)
Multi-nodular goiter	4	0 (0%)	0 (0%)

b. Other clinical supportive data: Not applicable
Clinical cut-off: 4.

Same as assay cut-off

5. Expected values/Reference range:

EliA anti-TG Immunoassay:

Antibody prevalence in autoimmune patients varies widely depending on disease area. A significant percentage (>10%) of healthy subjects was reported to have anti-thyroglobulin antibodies. Expected values may vary depending on the population tested.

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The 5th percentile of the tested normal samples was calculated as 5.4 IU/ml, the 95th percentile as 97.9 IU/ml.

EliA anti-TPO Immunoassay:

Antibody prevalence in autoimmune patients varies widely depending on disease area. A significant percentage (>10%) of healthy subjects was reported to have anti-thyroid peroxidase antibodies. Expected values may vary depending on the population tested.

The 5th percentile of the tested normal samples was calculated as 1.7 IU/ml, the 95th percentile as 59.9 IU/ml.

Regulation Number and Section

§ 866.5870 Thyroid autoantibody immunological test system.

(a)
Identification. A thyroid autoantibody immunological test system is a device that consists of the reagents used to measure by immunochemical techniques the thyroid autoantibodies (antibodies produced against the body's own tissues). Measurement of thyroid autoantibodies may aid in the diagnosis of certain thyroid disorders, such as Hashimoto's disease (chronic lymphocytic thyroiditis), nontoxic goiter (enlargement of thyroid gland), Grave's disease (enlargement of the thyroid gland with protrusion of the eyeballs), and cancer of the thyroid.(b)
Classification. Class II (performance standards).