The Thyretain ™ TSI Reporter BioAssay is intended for the qualitative detection in serum of thyroid stimulating autoantibodies to the thyroid stimulating hormone (TSH) receptors (TSHRs) on the thyroid. The detection of these stimulating autoantibodies, in conjunction with other clinical and laboratory findings, may be useful as an aid in the differential diagnosis of patients with Graves' disease (GD).

Device Description

The Thyretain ™ TSI Reporter BioAssay (TSI Reporter) utilizes a patented bioassay technology to detect thyroid stimulating immunoglobulin (TSI) in human serum. Genetically engineered Chinese hamster ovary (CHO) cells, expressing a chimeric form' of the human thyroid stimulating hormone receptor (TSHR) and a cyclic adenosine monophosphate (cAMP) induced luciferase reporter gene, are cryogenically preserved and provided in measured aliquots. The CHO Mc4 cell line has a nucleic acid sequence encoding a chimeric human TSH receptor, designed for reduced response to thyroid blocking immunoglobulin (TBI) activity. Thus, the hTSH receptor, comprised of 730 amino acids, has amino acid residues 262 to 335 replaced by the equivalently located 73 amino acid residues of the rat Luteinizing Hormone receptor to form the chimeric TSHR. This chimeric receptor is linked to a firefly luciferase reporter gene in operable combination with a glycoprotein hormone a-subunit promoter. The cells are seeded and grown for 15 to 18 hours to confluent monolayers in a 96-well plate. Patient sera, reference control, positive and normal controls and are diluted with a proprietary reaction buffer (RB), added to the cell monolayers and allowed to react with the cells for 3 hours. During this induction period TSI, if present in the patient serum, bind to the chimeric human TSHR on the cell surface. This binding event induces a signaling cascade resulting in increased production of intra-cellular cAMP. This increased production of cAMP is evidenced by increased production of luciferase. At the conclusion of the 3 hour induction period the cells are lysed. Luciferase levels are then measured using a luminometer. A significant increase in luminescence over the Reference Control indicates the presence of TSI antibodies in the sample.

AI/ML Overview

Here's a summary of the acceptance criteria and the studies that prove the device meets them, based on the provided text:

Thyretain™ TSI Reporter BioAssay: Acceptance Criteria and Study Summary

The provided document describes the Thyretain™ TSI Reporter BioAssay as an in-vitro diagnostic device intended for the qualitative detection of thyroid stimulating autoantibodies (TSI) in human serum, to aid in the differential diagnosis of Graves' disease (GD). The performance was assessed through non-clinical and clinical studies, primarily comparing it to a legally marketed predicate device, the KRONUS TSH Receptor Antibody (TRAb) Coated Tube (CT) Assay Kit.

1. Table of Acceptance Criteria and Reported Device Performance

The document doesn't explicitly state "acceptance criteria" for clinical performance in a numerical format that would typically precede a study. Instead, it presents the results of comparative studies against a predicate device and clinical sensitivity/specificity. The implied acceptance is that the device demonstrates comparable performance to the predicate and acceptable clinical utility (sensitivity and specificity).

Performance Measure	Acceptance Criteria (Implied by Comparison)	Reported Device Performance (Thyretain™ TSI Reporter BioAssay)
Non-Clinical Performance
Limit of Detection (LoD)	Not explicitly stated as a numerical criterion for acceptance, but demonstrated according to CLSI EP17A.	89.14% SRR
Interference	No observed interference (i.e., less than a defined threshold) from specified levels of bilirubin, hemoglobin, and lipids.	No interference observed with bilirubin up to 36.6 mg/dL, hemoglobin up to 250 mg/dL, and lipids up to 1,168 mg/dL.
Cross-reactivity (Glycoprotein Hormones)	No significant cross-reactivity (i.e., less than a defined threshold) with specified levels of other glycoprotein hormones.	No cross-reactivity observed with luteinizing hormone up to 625 mIU/mL, human chorionic gonadatrophin up to 40,625 mIU/mL, follicle stimulating hormone up to 2,000 mIU/mL, and thyroid stimulating hormone up to 0.35 mIU/mL.
Cross-reactivity (Other Autoantibodies)	Most samples with other autoimmune diseases should test negative for TSI.	1 out of 36 samples with autoimmune diseases (16 Hashimoto's, 10 RA, 10 SLE) tested positive (Hashimoto's sample with TSH levels near interference level). All other samples tested negative.
Intra-Assay Precision (CV%)	Implied: Low variability (e.g., typically ≤ 10-15% for immunoassays depending on the analyte level).	Average intra-plate (n=16) variation (CV %) was 4.7%.
Inter-Assay Precision (CV% - Intra-Day)	Implied: Low variability (e.g., typically ≤ 10-15% for immunoassays depending on the analyte level).	Average inter-assay CV% values (day one) were: High TSI (3.6%), Medium TSI (2.6%), Low TSI (4.2%), Reference Control (2.4%), TSI Positive Control (5.0%), Normal Control (5.0%). Overall inter-assay variation within this day was 3.8%.
Inter-Assay Precision (CV% - Inter-Day)	Implied: Acceptable long-term variability.	Overall average inter-assay variation across 20 days was 12% (individual sample types ranged from 7% to 16%).
Reproducibility (CV%)	For Samples A, B, C, D: Overall CV% for Sample A (23.7%), Sample B (23.7%), Sample C (24.6%), Sample D (17.9%). For Samples E, F, G: Overall CV% for Sample E (15.0%), Sample F (20.3%), Sample G (20.5%).
Reproducibility (Accuracy/Ratio)	For Samples A, B, C, D: Samples A & B: 100% positive ratio. Sample C: 100% negative ratio. Sample D: 50% positive ratio (139/180). For Samples E, F, G: Sample E: 100% positive ratio (60/60). Samples F & G: 100% negative ratio (60/60). Note: The acceptance criteria were defined as "Expected Accuracy" of 100% for A, B, C, E, F, G and 50% for D, and the device met these except for Sample D which was 139/180 positive, which is close to the 50% target.

Clinical Performance		Reported Device Performance (Thyretain™ TSI Reporter BioAssay)
Comparative Study vs. Predicate	"Comparable" to the predicate device (KRONUS TRAb). No specific numerical thresholds are provided for PPA/NPA.	Combined Sites 1-COH and 2-MN (n=299 valid specimens):- Positive Percent Agreement (PPA): 93.8% (95% CI: 88.2% to 96.8%)- Negative Percent Agreement (NPA): 89.5% (95% CI: 84.0% to 93.2%)Site 3-NC (n=231 valid specimens):- Positive Percent Agreement: 74.6% (95% CI: 63.5% to 83.3%)- Negative Percent Agreement: 97.5% (95% CI: 93.8% to 99.0%)Post-hoc analysis removing hypothyroid patients at Site 3-NC increased PPA to 81.5% (95% CI: 70.4% to 89.1%).
Clinical Sensitivity/Specificity	Not explicitly stated as a numerical criterion for acceptance, but demonstrated to provide useful diagnostic information.	Study with 249 characterized specimens:- Clinical Sensitivity: 92.0% (46/50 Graves Disease positive)- Clinical Specificity: 99.5% (198/199 Other autoimmune diseases and healthy controls negative)

2. Sample Sizes and Data Provenance

Non-Clinical Studies:
- Assay Cutoff:
  - "Training set": 30 subjects with diagnosed Graves' disease and 44 normal subjects.
  - "Testing-set" (pre-clinical verification): 50 GD positive sera, 140 normal sera.
- Precision (Intra-assay, Inter-assay, Inter-Day): Varies per test; for Inter-Day, n=120 for high, medium, low TSI serum, n=40 for normal serum, n=80 for controls.
- Reproducibility:
  - Panel 1: 4 specimens tested at multiple sites (3 sites, with one having 2 technicians). Each site/technician performed testing twice a day over 8 days, leading to 180 total tests for each sample (3 sites x 2 tests/day x 8 days x 4 samples = 192, or 3 sites x 2 techs x 2 tests/day x 8 days = 96 for specific site/tech data, actual calculation seems to be 180 total for "positive ratio").
  - Panel 2: 3 samples tested at 2 sites twice a day for 5 days, totaling 60 tests for each sample (2 sites x 2 tests/day x 5 days x 3 samples = 60).
Clinical Performance Studies:
- Comparative Study:
  - Combined Sites 1-COH and 2-MN: 312 specimens initially, 299 analyzed (1 excluded for insufficient quantity, 12 excluded due to indeterminate results on comparator).
  - Site 3-NC: 247 specimens initially, 231 analyzed (16 excluded due to indeterminate results on comparator).
- Clinical Sensitivity and Specificity: 249 characterized specimens.
Data Provenance: The document implies the data is retrospective/archived samples, as it refers to "patients with diagnosed Graves' disease," "normal subjects with no known or clinically diagnosed thyroid disease," and "sera obtained from physicians with diagnostic information." The multi-site nature (COH, MN, NC) suggests geographical diversity within the US. There is no explicit mention of the country of origin for all samples.

3. Number of Experts and Qualifications for Ground Truth

Non-Clinical Studies (Assay Cutoff): Ground truth was established based on "diagnosed Graves' disease" and "normal subjects with no known or clinically diagnosed thyroid disease," implied to be clinical diagnosis by physicians. No specific number or qualifications of experts are provided.
Clinical Performance Studies (Comparative Study, Clinical Sensitivity/Specificity):
- The "ground truth" for the comparative study was the results from the KRONUS TSH Receptor Antibody (TRAb) Coated Tube (CT) Assay Kit (the predicate device). This implies that the predicate device's results were accepted as the reference for comparison against the subject device.
- For the Clinical Sensitivity and Specificity study, specimens were "249 characterized specimens" categorized as "Graves Disease" and "Other autoimmune diseases and healthy controls." This categorization itself acts as the ground truth. The method of characterization (e.g., expert clinical diagnosis, pathology) is not explicitly detailed, but it is implied to be based on clinical diagnosis ("Diagnosis"). No specific number of experts or their qualifications are provided for establishing these fundamental diagnoses.

4. Adjudication Method

The document does not describe an adjudication method for establishing ground truth using multiple experts.
For the comparative study, discordant results between the subject device and the predicate device were analyzed, especially at Site 3-NC. The analysis focused on patient TSH results and ATA guidelines for hypothyroidism to explain the discrepancies, rather than an expert adjudication of the initial diagnosis. No multi-reader, observer, or expert consensus adjudication is described for the ground truth of the patient samples.

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

No MRMC comparative effectiveness study is described where human readers' performance with and without AI assistance is evaluated. The device is an in-vitro diagnostic assay read by a luminometer, not an AI interpreting images for human readers.

6. Standalone Performance

Yes, the clinical performance studies (both the comparative study and the clinical sensitivity/specificity study) describe the standalone performance of the Thyretain™ TSI Reporter BioAssay. The device produced its own results (positive/negative) which were then compared to either the predicate device's results or defined clinical diagnoses.

7. Type of Ground Truth Used

Non-Clinical (Assay Cutoff): Clinical diagnosis of Graves' disease or no known thyroid disease.
Clinical Performance (Comparative Study): The results of the predicate device (KRONUS TSH Receptor Antibody (TRAb) Coated Tube (CT) Assay Kit) were used as the reference for comparison.
Clinical Performance (Clinical Sensitivity and Specificity): Clinical diagnosis ("Graves Disease", "Other autoimmune diseases and healthy controls"). This would typically be established based on a combination of clinical signs/symptoms, other laboratory tests, and possibly imaging, by a physician.

8. Sample Size for the Training Set

For establishing the preliminary assay cutoff:
- 30 subjects with diagnosed Graves' disease.
- 44 normal subjects with no known or clinically diagnosed thyroid disease.
- (An additional "testing-set" of 50 GD positive sera and 140 normal sera was used for verification).

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

The ground truth for the training set (for assay cutoff determination) was established through clinical diagnosis: "subjects with diagnosed Graves' disease" and "normal subjects with no known or clinically diagnosed thyroid disease." This implies traditional clinical assessment by physicians.

Summary

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KC83391

Thyretain™ TSI Reporter BioAssay SECTION 05, 510(K) SUMMARY

Image /page/0/Picture/2 description: The image shows the logo for Diagnostic Hybrids. The logo features the company name in bold, sans-serif font, with the word "DIAGNOSTIC" stacked above "HYBRIDS." Below the company name, the tagline "Integrating Science and Humanity" is displayed in a smaller, italicized font. To the right of the text, there is a stylized graphic of a human figure intertwined with a DNA double helix.

APPLICANT

DIAGNOSTIC HYBRIDS, INC. 1055 East State Street Suite 100 Athens, OHIO 45701

MAY 21 2009

CONTACT INFORMATION

Ronald H. Lollar Senior Director, Product Realization, Management, and Marketing E-mail: lollar@dhiusa.com Telephone: 740-589-3300 Desk Extension: 740-589-3373 FAX: 740-593-8437

DATE OF PREPARATION OF 510(k) SUMMARY

November 14, 2008

DEVICE NAME

Trade name: Thyretain TM TSI Reporter BioAssay Common name: TSI Reporter Classification name: System, Test, Thyroid Autoantibody Product Code: JZO Regulation: 21 CFR § 866.5870, Thyroid Autoantibody Immunological Test

DEVICE DESCRIPTION

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The cells are seeded and grown for 15 to 18 hours to confluent monolayers in a 96-well plate. Patient sera, reference control, positive and normal controls and are diluted with a proprietary reaction buffer (RB), added to the cell monolayers and allowed to react with the cells for 3 hours. During this induction period TSI, if present in the patient serum, bind to the chimeric human TSHR on the cell surface. This binding event induces a signaling cascade resulting in increased production of intra-cellular cAMP. This increased production of cAMP is evidenced by increased production of luciferase. At the conclusion of the 3 hour induction period the cells are lysed. Luciferase levels are then measured using a luminometer. A significant increase in luminescence over the Reference Control indicates the presence of TSI antibodies in the sample.

KIT COMPONENTS

1. CHO Mc4 FreshFrozenCells®: Cryovials containing CHO Mc4 cells cryogenically preserved in cryoprotective medium containing DMSO. Reagent is stored at -70°C or lower.
1. Cell Attachment Solution, 100-mL: A proprietary reagent used to treat the wells of a 96-well plate prior to planting the cells that promotes rapid cell attachment. Reagent is stored at 15° to 30°C.
1. Growth Medium, 100-mL: Hamm's F-12 cell culture medium containing 10% FBS. Reagent is stored at 20 to 8ºC.
1. Reaction Buffer, 500-mL: A proprietary buffer that augments the reaction of TSI with the TSHR. Reagent is stored at 2º to 8ºC.
1. Control Set:
- Positive Control, 0.5-mL: TSI-containing human serum which yields a value a. that is ≥140% of the Reference Control. Reagent is stored at -70℃ or lower.
- Reference Control, 0.5-mL: A bTSH-containing solution against which b. Controls and Test Specimens are compared. Reagent is stored at -70°C or lower.
- c. Normal Control, 0.5-mL: Human serum that is negative for the presence of TSI which yields a value that is <140% of the Reference Control. Reagent is stored at -70°C or lower.
1. Luciferase Assay Reagent Set:
- a. Luciferase Substrate, 1 vial: A lyophylized beetle luciferin substrate which is converted by luciferase to oxyluciferin and light. Reagent is stored at -20°C or lower.
- b. Luciferase Assay Buffer Solution, 1 vial, 10-mL: A cell culture lysis buffer.

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INTENDED USE

Legally marketed device to which equivalence is claimed:

KRONUS TSH Receptor Antibody (TRAb) Coated Tube (CT) Assay Kit k()32134

Intended Use: The KRONUS TSH Receptor Antibody (TRAb) Coated Tube (CT) Assay Kit is designed to measure human serum autoantibodies to the thyroid stimulating hormone (TSH or thyrotropin) receptor. The TRAb CT kit is useful as an aid in the differential diagnosis of Graves' Disease.

Table 5.1: Subject Device and Predicate Device Characteristics
Similarities
Item	Subject Device	Predicate Device
Intended Use	The ThyretainTM TSI ReporterBioAssay is intended for thequalitative detection of thyroidstimulating autoantibodies to thethyroid stimulating hormonesreceptors (TSHRs) on thethyroid. The detection of thesestimulating autoantibodies, inconjunction with other clinicaland laboratory findings, may beuseful as an aid in thedifferential diagnosis of patientswith Graves' disease.	KRONUS TSDH ReceptorAntibody (TRAb) Coated Tube(CT) Assay Kit is designed tomeasure human serumautoantibodies to the thyroidstimulating hormone (TSH orthyrotropin) receptor. The TRAbCT kit is useful as an aid in thedifferential diagnosis of Graves'disease.
Sample matrix	Serum	same
Differences
Item	Subject Device	Predicate Device
Trade name	ThyretainTM TSI ReporterBioAssay	KRONUS TSH ReceptorAntibody (TRAb) Coated Tube(CT) Assay Kit
Assay Format	Qualitative	Qualitative and quantitative
Assay principle	Cell-based Chemiluminescent	Radioreceptor Assay

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Table 5.1: Subject Device and Predicate Device Characteristics
Differences
Item	Subject Device	Predicate Device
Solid Phase	CHO Mc4 cell monolayer(96-well microplate)	TSHR-coated tube
Analyte	Thyroid StimulatingImmunoglobulin to Thyroidstimulating hormone (TSI-hyperthyroidism)	TSHR antibodies including bothThyroid stimulating hormone(TSI-hyperthyroidism) andblocking hormone (TBI-hypothyroidism)
Calibration	NIBSC Standard 03/192 orsimilar standard	NIBSC Standard 90/672 orsimilar calibrator
Signal	Relative Light Units	Radioactivity
Detectioninstrument	Luminometer	Gamma counter set for 125I
Cut-off	Positive: ≥ 140% of Reference ControlNegative: < 140% of Reference Control	Positive: 15% inhibitionIndeterminate: 11-15% inhibitionNegative: <11% inhibition

NON-CLINICAL PERFORMANCE

1. Limit of Detection

The LoD for the TSI Reporter has been determined to be 89.14 %SRR based on the calculations presented in CLSI EP17A.

2. Interference by Endogenous Substances

Interference was not observed in TSI-containing human serum that was spiked with

· bilirubin up to 36.6 mg/dL,
hemoglobin up to 250 mg/dL and .
. lipids up to 1,168 mg/dL.

3. Cross-reactivity by Glycoprotein Hormones

luteinizing hormone up to 625 mIU/mL, .
human chorionic gonadatrophin up to 40,625 mIU/mL, .
follicle stimulating hormone up to 2,000 mIU/mL, .
thyroid stimulating hormone up to 0.35 mIU/mL.

4. Cross-reactivity with Other Autoantibodies

The TSI Reporter was tested on 36 samples with autoimmune diseases other than GD:

· 16 autoimmune hypothyroidism (Hashimotos' disease [Hm]),
· 10 Rheumatoid Arthritis (RA) and
· 10 Systemic Lupus Erythematosus (SLE).

One (1) Hm sample tested positive for TSI, however, this is a sample with TSH levels at or near the level of interference reported above. All other samples tested negative.

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5. Assay Cutoff

Patient serum, when tested using the TSI Reporter, will be considered positive for the presence of TSI if the resultant SRR% measures greater than or equal to (≥) 140% over the Reference Control. This is a preliminary cut-off which was established using a . "training-set" of samples and was confirmed in the clinical studies. The TSI Reporter prototype device was tested for assay cutoff limits through testing of 30 subjects with diagnosed Graves' disease and 44 normal subjects with no known or clinically diagnosed thyroid disease. The SRR% data obtained for each of these subjects were analyzed using receiver operating characteristic (ROC) curve analysis.

Using a "testing-set" of samples, the preliminary cutoff was verified in pre-clinical testing with an additional 50 GD positive sera obtained from physicians with diagnostic information and 140 normal sera. Additional verification of the cutoff limit was performed through precision testing. A manufactured sample near the cutoff limit (~ 157% above the Reference Control) was tested over 20 days (n=120) and yielded positive results ≥75% of the time and negative results ≤25% of the time. CLSI recommendations for assay cutoff verification (Evaluation Protocol (EP) 12-A, Section 7.0) are a 50%/50% positive to negative ratio for samples at the cutoff.

6. Intra-Assay Precision

The TSI Reporter was conducted by a single user over a 20 day period following the Precision Study Protocol (see Protocols and Procedures). Each sample was tested in triplicate wells and the resulting RLU values were averaged per sample. Each plate of cells analyzed per day contained 16 samples. The average variation for each sample was calculated (CV %) across each plate. It was calculated that the average intra-plate (n=16) variation (CV %) was 4.7%.

7. Inter-Assay Precision (Intra-Day)

The same samples tested during the Precision Study were analyzed for inter-assay precision within each day. Serum samples were manufactured to simulate a patient with high levels, medium levels and low levels of TSI by diluting serum from a known GD subject with highly stimulatory TSI into normal human serum and tested repeatedly. Serum from a TSI negative subject, TSI Positive Control and Reference Control were also tested. The total replicates for the types of samples tested per day were 6, 6, 6, 6, 2, and 4 for the high positive, medium positive, low positive, normal, TSI Positive Control and Reference Control respectively. The average triplicate was calculated per plate then analyzed for inter-plate variation. For the high TSI, medium TSI and low TSI containing serum, the average inter-assay CV% values calculated on day one of precision testing were 3.6%, 2.6% and 4.2% respectively. The Reference Control, TSI Positive Control and Normal Control were 2.4%, 5.0% and 5.0% respectively, with an overall inter-assay variation within this day calculated to be 3.8%.

8. Inter-Assay Precision (Inter-Day)

The data collected from the above studies were analyzed for inter-assay precision across a 20 day study. When combined, the average variation of the assay for the high TSI

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containing serum, medium TSI containing serum, low TSI containing serum, normal serum, TSI Positive Control and Reference Control (n=120, n=120, n=120, n=40 and n=80) was 12%, 13%, 16%, 12%, 12% and 7% respectively, with an overall average inter-assay variation across 20 days calculated to be 12%.

9. Assay Reproducibility

To demonstrate competency with and reproducibility of the TSI Reporter, each trained site performed testing on the panel® described in Table 5.2 below twice a day over an eight day span:

Table 5.2: Reproducibility Panel Variation and Accuracy
Reagent	Volume	ExpectedVariation	Expected Accuracy
Specimen A	15x500 µL	+/- 15%	100%
Specimen B	15x500 µL	+/- 15%	100%
Specimen C	15x500 µL	+/- 25%	100%
Specimen D	15x500 µL	+/- 15%	50%

Table 5.3 reports the results for each of the sites over their proficiency training period. Site 3 requested 2 technicians be trained

Table 5.3: Proficiency Training Period Site Results (reported as Average SRR%and Coefficient of Variation)
Site 1-COH	Site 2 - MN	Site 3 - NCTechnician 1	Site 3 - NCTechnician 2
Sample A	Sample A	Sample A	Sample A
270.09% 11.47%	439.89% 10.51%	280.10% 13.56%	289.49% 10.55%
Sample B	Sample B	Sample B	Sample B
293.11% 14.72%	495.14% 14.59%	374.26% 14.70%	345.13% 11.80%
Sample C	Sample C	Sample C	Sample C
48.23% 14.94%	67.89% 14.66%	44.33% 20.21%	48.60% 22.46%
Sample D	Sample D	Sample D	Sample D
158.6% 10.62%	198.0% 10.96%	143.6% 14.43%	142.7% 12.12%

All sites performed the study using manufactured panel samples. Each site's data was analyzed cumulatively to determine the Reproducibility and Repeatability of the panel samples. Samples A and B both had a positive ratio (Number Positive/Total Number

a The panel consisted of four specimens created to meet the requirements for precision as set forth in both CLSI (EP12-A2) and FDA guidance documents (http://www.fda.gov/cdrh/ode/odec1051.html)

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Tested) of 180/180, Sample C had a negative ratio (Number Negative/Total Number Tested) of 180/180 and Sample D had a positive ratio (Number Positive/Total Number Tested) of 139/180. The overall coefficient of variation (CV) % for Samples A, B, C and D were 23.7%, 23.7%, 24.6% and 17.9% respectively.

An additional smaller study was performed using three samples near the cut off was performed at two sites twice a day for five-days.

Table 5.4: Reproducibility Panel 2 Variation and Accuracy
Reagent	Volume	ExpectedVariation	Expected Accuracy
Specimen E	15x500 µL	+/- 15%	100%
Specimen F	15x500 µL	+/- 25%	100%
Specimen G	15x500 µL	+/- 25%	100%

Table 5.5 reports the results for each of the two sites over their proficiency training period.

Table 5.5: Reproducibility Results (reported as Average SRR% and Coefficient of Variation)
	2-MN	Diagnostic Hybrids Inc.
Specimen E	194%9.3%	170%17.7%
Specimen F	106%20.2%	106%20.7%
Specimen G	102%21.6%	107%19.4%

Each site's data was analyzed cumulatively to determine the Reproducibility and Repeatability of the panel samples. Sample E had a positive ratio (Number Positive/Total Number Tested) of 60/60, Samples F and G had negative ratios (Number Negative/Total Number Tested) of 60/60. The overall coefficient of variation (CV) % for Samples E, F and G were 15.0%, 20.3%, and 20.5% respectively.

10. Assay Stability

Stability was determined through repeated testing of the assay over three lots of materials. All assay studies are carried out according to the final written procedure found in the Instructions for Use. Assay results were evaluated as a ratio of the sample over the reference control and are compared to each previous data set throughout the

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study. The data has been trended over time. The variation of the results [including standard deviations and coefficient of variation (%)] were reported over time along with the sample result. The TSI Reporter materials, reagents and components are stable and pass all stipulated acceptance criteria for a minimum of 12 months.

CLINICAL PERFORMANCE

Studies began at two testing sites with a total of 312 specimens to be evaluated by both the subject and comparator devices. One specimen was excluded due to insufficient quantity for testing. Twelve of these specimens were excluded from statistical analysis due to indeterminate results on the comparator device. The remaining 299 specimens were analyzed for positive and negative percent agreement.

The following table (Table 5.6) details summary results from study site 1-COH and 2-MN combined information.

Table 5.6: Sites 1-COH and 2-MN Combined Result Summary
299 specimen results	Comparator Device (KRONUS TRAb)
	+	-	Indeterminate
Subject Device(TSI Reporter)	+	120	18	7
	-	8	153	5
		95% Confidence Interval
Positive Percent Agreement	93.8%	88.2 to 96.8%
Negative Percent Agreement	89.5%	84.0 to 93.2%

The ability of the subject device to detect TSI using a cell-based system was compared to the comparator device's radioreceptor assay. The positive percent agreement for study sites 1-COH and 2-MN combined was 93.8% (95% CI range 88.2% to 96.8%). The negative percent agreement for study sites 1-COH and 2-MN combined was 89.5% (95% Cl range 84.0% to 93.2%). The performance data for the two devices are comparable and, therefore it appears that the TSI Reporter is substantially equivalent to the KRONUS TRAb.

An additional study was performed at a third testing site using 247 specimens. Sixteen of these specimens were excluded from statistical analysis due to indeterminate results on the comparator device. The remaining 231 specimens were analyzed for positive and negative percent agreement.

The following table (Table 5.7) details summary results from study site 3-NC.

Table 5.7: Site 3-NC Result Summary
231 specimen results
	Comparator Device (KRONUS TRAb)
	+	-	Indeterminate
Subject Device(TSI Reporter)	+	53	4	2
	-	18	156	14
	95% Confidence Interval

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Positive Percent Agreement	74.6%	63.5 to 83.3%
Negative Percent Agreement	97.5%	93.8 to 99.0%

The positive percent agreement was 74.6% (95% CI range 63.5% to 83.3%). The negative percent agreement was 97.5% (95% Cl range 93.8% to 99.0%). An explanation for this decreased positive percent agreement value is the specificity difference between TSI Reporter and the comparator device. The comparator device detects autoantibodies to the TSHR, of which there are two classes, stimulating (TSI-hyperthyroidism) and blocking (TBI-hypothyroidism). The comparator device is unable to distinguish between the two antibody types, as stated in the Assay Limitations of the comparator device's product insert. TSI Reporter detects only TSI. Thus, during our clinical performance testing, it is likely that some patient sera have TBI and would be positive by the comparator device but negative by the subject device. There currently is no TBI specific cleared device and, as such, it is not possible to further analyze the discordant results. There is no published data indicating the prevalence of TBI in the normal population.

The patient TSH results from the 18 discordant specimens (Comparator Device positive/Subject Device negative) at study site 3-NC were further reviewed. The results indicate that 6 patients meet the ATA definition of hypothyroidism (>3.0). If these patients are removed from the dataset from site 3 the PPA increases to 81.5% (95% CI range 70.4% to 89.1%).

Study Site 3-NC is a reference laboratory that receives specimens from all medical disciplines to be tested as part of a thyroid screening panel. The testing of patients with hypothyroidism is more likely in a screening environment than at Sites 1-COH and 2-MN that service targeted disciplines (i.e. Endocrinologists and Thyroidologists). ATA guidelines defines Hypothyroidism as a high TSH level (>3.0). A review of the TSH levels from the three sites indicate a greater percentage of Hypothyroid patients were in fact evaluated at Study Site 3-NC (24% of specimens) than at Study Sites 1-COH and 2-MN (15% and 11%, respectively).

There was good correlation between the three study sites regarding negative percent agreement.

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CLINICAL SENSITIVITY AND SPECIFICITY

The Clinical Sensitivity and Specificity for the device was determined by testing 249 characterized specimens. The data is presented in the table 5.8 below.

		Table 5.8: Clinical Sensitivity and Specificity
		Diagnosis
		Positives(Graves Disease)	Negative(Other autoimmunediseases and healthycontrols)	Totals
TSIReporter	Positive	46	1	47
	Negative	4	198	202
	Total	50	199

Clinical Sensitivity: 92.0% (46/50) Clinical Specificity: 99.5% (198/199)

CONCLUSION

Evidence presented in the above discussion supports substantial equivalence of the subject device, The Thyretain ™ TSI Reporter BioAssay (TSI Reporter), to KRONUS TSH Receptor Antibody (TRAb) Coated Tube (CT) Assay Kit, a device regulated under 21 CFR $ 866.5870, product code JZO.

l Tahara K., Ban T., Minegishi T., Kohn L.D. Immunoglobulins from Graves' disease patients interact with different sites on TSH receptor/LH-CG receptor chimeras than either TSH or immunoglobulins from idiopathic myxedema patients. Biochem Biophys Res Commun. 1991 Aug 30; 179(1):70-7

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DEPARTMENT OF HEALTH & HUMAN SERVICES

Image /page/10/Picture/1 description: The image shows the logo for the U.S. Department of Health & Human Services. The logo features a stylized eagle with three tail feathers, representing the department's mission to protect the health of all Americans and provide essential human services. The eagle is encircled by the words "DEPARTMENT OF HEALTH & HUMAN SERVICES. USA" in a circular arrangement.

Public Health Service

Food and Drug Administration 2098 Gaither Road Rockville MD 20850

MAY 21 2009

DIAGNOSTIC HYBRIDS Inc c/o Ms. Gail R. Goodrum Vice President of Regulatory Affairs 1055 East State Street Suite 100. Athens, OH 45701

Re: K083391

Trade/Device Name: Thyretain™ TSI Reporter BioAssay Regulation Number: 21 CFR §866.5870 Regulation Name: Thyroid autoantibody immunological test system Regulatory Class: Class II Product Code: JZO Dated: May 7, 2009 Received: May 08, 2009

Dear Ms. Goodrum:

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.

If your device is classified (see above) into either class II (Special Controls) or class III (PMA), it may be subject to such additional controls. Existing major regulations affecting your device can be found in Title 21, Code of Federal Regulations (CFR), Parts 800 to 895. 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); and good manufacturing practice requirements as set forth in the quality systems (QS) regulation (21 CFR Part 820). This letter

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

If you desire specific advice for your device on our labeling regulation (21 CFR Part 801), please contact the Office of In Vitro Diagnostic Device Evaluation and Safety at (240) 276-0450. Also, please note the regulation entitled, "Misbranding by reference to premarket notification" (21 CFR Part 807.97). For questions regarding postmarket surveillance, please contact CDRH's Office of Surveillance and Biometric's (OSB's) Division of Postmarket Surveillance at (240) 276-3474. For questions regarding the reporting of device adverse events (Medical Device Reporting (MDR)), please contact the Division of Surveillance Systems at (240) 276-3464. For more information regarding the reporting of adverse events, please go to http://www.fda.gov/cdrh/mdr/.

You may obtain other general information on your responsibilities under the Act from the Division of Small Manufacturers, International and Consumer Assistance at its toll-free number (800) 638-2041 or (240) 276-3150 or at its Internet address http://www.fda.gov/cdrh/industry/support/index.html.

Sincerely vours,

Maria M Chan

Maria M. Chan, Ph.D. Director Division of Immunology and Hematology Devices Office of In Vitro Diagnostic Device Evaluation and Safety Center for Devices and Radiological Health

Enclosure

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Indication for Use

510(k) Number (if known): K083391

Device Name: Thyretain ™ TSI Reporter BioAssay

Indication For Use:

Prescription Use ﺮ (21 CFR Part 801 Subpart D)

And/Or

Over the Counter Use (21 CFR Part 801 Subpart C)

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

Concurrence of CDRH, Office of In Vitro Diagnostic Device Evaluation and Safety (OIVD)

Munai m. Chen

Division Sign-Off Office of In Vitro Diagnostic Device Evaluation and Safety .

510(k) _k085391/

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).