ELECSYS FT4 II ASSAY, ELECSYS FT4 II CALSET

K131244 · Roche Diagnostics · CEC · Jan 27, 2014 · Clinical Chemistry

Device Facts

• Record ID: K131244
• Device Name: ELECSYS FT4 II ASSAY, ELECSYS FT4 II CALSET
• Applicant: Roche Diagnostics
• Product Code: CEC · Clinical Chemistry
• Decision Date: Jan 27, 2014
• Decision: SESE
• Submission Type: Traditional
• Regulation: 21 CFR 862.1695
• Device Class: Class 2
• Attributes: Real-World Evidence

Real-World Evidence

Submission	Device	Sponsor	RWD Sources	RWE Use Summary	Key Tags
K131244 · Jan 27, 2014	ELECSYS FT4 II ASSAY, ELECSYS FT4 II CALSET	Roche Diagnostics	Remnant clinical samples	Remnant clinical samples were used in a method comparison study to evaluate the performance of the Elecsys FT4 II assay against the predicate device.	Method comparison; Remnant clinical samples

Clinical Evidence

Study Design	Population	Comparator	Key Endpoints
Method comparison study	170 human serum samples; Sample Size: 170	Elecsys FT4 assay (predicate)	Passing/Bablok and Linear Regression analysis; bias at medical decision points

Indications for Use

The Elecsys FT4 II assay is for the in vitro quantitative determination of free Thyroxine in human serum and plasma. Measurements obtained by this device are used in the diagnosis and treatment of thyroid diseases. The electrochemiluminescence immunoassay "ECLIA" is intended for use on Elecsys and cobas e immunoassay analyzers. The Elecsys FT4 II CalSet is used for calibrating the quantitative Elecsys FT4 II assay on the Elecsys and cobas e immunoassay analyzers.

Device Story

The Elecsys FT4 II Assay is a two-step competitive immunoassay used for the quantitative determination of free thyroxine (FT4) in human serum and plasma. The device utilizes streptavidin-coated microparticles, a T4-specific polyclonal anti-T4 antibody (sheep) labeled with a sulfonyl-ruthenium complex, and electrochemiluminescence (ECLIA) detection. The assay is performed on automated Elecsys and cobas e immunoassay analyzers. The system requires 15 µL of sample and has a total assay duration of 18 minutes. Results are calculated via a master curve stored on the reagent barcode, adjusted by a two-point calibration using the Elecsys FT4 II CalSet. The output is a quantitative concentration of FT4, which clinicians use to diagnose and monitor thyroid diseases. The device is intended for professional use in clinical laboratory settings.

Clinical Evidence

Bench testing only. Performance evaluated per CLSI guidelines (EP5-A2, EP17-A2, EP6-A, C28-A3c). Precision studies (n=84) showed repeatability CVs of 0.9-4.0% and intermediate precision CVs of 1.8-7.6%. LoB ≤ 0.03 ng/dL, LoD ≤ 0.05 ng/dL, and LoQ 0.1 ng/dL. Linearity confirmed from 0.096 to 8.20 ng/dL. Method comparison (n=170) against the predicate showed Passing/Bablok slope 1.00 ± 0.05 and correlation r ≥ 0.95. Reference range validation (n=60) confirmed the established range of 0.93-1.7 ng/dL.

Technological Characteristics

Competitive immunoassay using electrochemiluminescence (ECLIA). Reagents: streptavidin-coated microparticles, sheep polyclonal anti-T4 antibody labeled with sulfonyl-ruthenium complex. Sample volume: 15 µL. Calibration: two-point, master curve via barcode. Platform: automated Elecsys and cobas e immunoassay analyzers. Stability: 84 days opened at 2-8°C; 28 days onboard. Sterilization: N/A (reagent kit).

Indications for Use

Indicated for in vitro quantitative determination of free Thyroxine in human serum and plasma to aid in the diagnosis and treatment of thyroid diseases. For use by clinical laboratory professionals on Elecsys and cobas e immunoassay analyzers.

Regulatory Classification

Identification

A free thyroxine test system is a device intended to measure free (not protein bound) thyroxine (thyroid hormone) in serum or plasma. Levels of free thyroxine in plasma are thought to reflect the amount of thyroxine hormone available to the cells and may therefore determine the clinical metabolic status of thyroxine. Measurements obtained by this device are used in the diagnosis and treatment of thyroid diseases.

Predicate Devices

• Elecsys FT4 Assay (k961489)
• Elecsys FT4 CalSet (k961489)

Submission Summary (Full Text)

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510(k) SUBSTANTIAL EQUIVALENCE DETERMINATION
DECISION SUMMARY
ASSAY ONLY TEMPLATE

A. 510(k) Number:
k131244

B. Purpose for Submission:
Modification of a previously cleared assay (k961489)

C. Measurand:
Free thyroxine (Free T4)

D. Type of Test:
Quantitative, electrochemiluminescent immunoassay

E. Applicant:
Roche Diagnostics

F. Proprietary and Established Names:
Elecsys FT4 II Assay
Elecsys FT4 II CalSet

G. Regulatory Information:
1. Regulation section:
21 CFR 862.1695
21 CFR 862.1150
2. Classification:
Both Class II
3. Product code:
CEC
JIT

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4. Panel:
Clinical Chemistry (75)

H. Intended Use:

1. Intended use(s):
Refer to Indications for Use below

2. Indication(s) for use:
The Elecsys FT4 II assay is for the in vitro quantitative determination of free Thyroxine in human serum and plasma. Measurements obtained by this device are used in the diagnosis and treatment of thyroid diseases.

The electrochemiluminescence immunoassay "ECLIA" is intended for use on Elecsys and cobas e immunoassay analyzers

The Elecsys FT4 II CalSet is used for calibrating the quantitative Elecsys FT4 II assay on the Elecsys and cobas e immunoassay analyzers.

3. Special conditions for use statement(s):
For in vitro diagnostic use only
For prescription use only

4. Special instrument requirements:
Roche Cobas e411 Analyzer only

I. Device Description:

The assay contains the following components:

M: Streptavidin-coated microparticles (transparent cap), 1 bottle, 12 mL: Streptavidin-coated microparticles 0.72 mg/mL; preservative.

R1: Anti-T4-Ab~Ru(bpy) (gray cap), 1 bottle, 18 mL: Polyclonal anti-T4-antibody (sheep) labeled with ruthenium complex 75 ng/mL; phosphate buffer 100 mmol/L, pH 7.0; preservative.

R2: T4~biotin (black cap), 1 bottle, 18 mL: Biotinylated T4 2.5 ng/mL; phosphate buffer 100 mmol/L, pH 7.0; preservative.

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J. Substantial Equivalence Information:

1. Predicate device name(s):
Elecsys FT4 Assay, Elecsys FT4 CalSet

2. Predicate 510(k) number:
k961489

3. Comparison with predicate:
Elecsys FT4 II assay

| Similarities | | |
| --- | --- | --- |
| Item | New Device (reagent) | Predicate |
| Intended Use | Quantitative determination of free thyroxine (FT4) in serum and plasma | Same |
| Methodology | Quantitative electrochemiluminescence immunoassay | Same |
| Reference Range | Euthyroid: 0.93–1.7 ng/dL | Same |
| Sample Volume | 15 μL | Same |
| Calibrators | 2 levels | Same |
| Assay Time | 18 minutes | Same |
| Differences | | |
| --- | --- | --- |
| Item | New Device (reagent) | Predicate |
| Measuring Range | 0.1 – 7.77 ng/mL | 0.02 – 7.77 ng/mL |
| Acceptable Sample Matrices | Serum
Plasma anticoagulated with K^{2} EDTA, K^{3} EDTA, and Lithium Heparin | Serum
Plasma anticoagulated with K^{3} EDTA, Lithium Heparin, Sodium Heparin, NH4+ Heparin, Sodium Citrate, Sodium Fluoride/Potassium Oxalate |
| Traceability/Standardization | Standardized against the Elecsys FT4 method. The Elecsys FT4 assay is traceable to the Enzymun-Test which was standardized using equilibrium dialysis. | Standardized against the Enzymun-Test FT4 method, which was standardized using equilibrium dialysis. |

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Elecsys FT4 II CalSet

| Similarities | | |
| --- | --- | --- |
| Item | New Device (calibrators) | Predicate |
| Intended Use | Intended for the calibration of Roche Free T4 Immunoassay | Same |
| Calibrator concentrations (exact calibrator concentration is lot-specific) | Cal 1: Approximately 0.78 ng/dL | Same |
| | Cal 2: Approximately 3.5 ng/dL | |
| Opened stability at 2 – 8°C | 12 weeks | Same |
| Differences | | |
| --- | --- | --- |
| Item | New Device (calibrators) | Predicate |
| Name | Elecsys FT4 II CalSet | Elecsys FT4 CalSet |

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

CLSI EP5-A2: Evaluation of Precision Performance of Quantitative Measurement Methods: Approved Guideline, Second Edition

CLSI EP 6-A: Evaluation of the Linearity of Quantitative Measurement Procedures: A statistical Approach: Approved Guideline

CLSI EP 17-A2, Evaluation of Detection Capability for Clinical Laboratory Measurement Procedures: Approved Guideline, Second Edition

## L. Test Principle:

This is a competitive assay with an assay time of 18 minutes.

First incubation: 15 µL of sample is combined with a T4-specific antibody labeled with a sulfonyl-ruthenium complex.

Second incubation: After addition of biotinylated T4 and streptavidin-coated microparticles, the still-free binding sites of the labeled antibody become occupied, with formation of an antibody-hapten complex. The entire complex becomes bound to the solid phase via interaction of biotin and streptavidin.

The reaction mixture is then aspirated into the measuring cell where the microparticles are magnetically captured onto the surface of the electrode. Unbound substances are then removed with ProCell/ProCell M. Application of a voltage to the electrode then induces

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chemiluminescent emission which is measured by a photomultiplier.

Results are determined via a calibration curve which is instrument-specifically generated by 2-point calibration and a master curve provided via the reagent barcode.

## M. Performance Characteristics (if/when applicable):

### 1. Analytical performance:

#### a. Precision/Reproducibility:

The repeatability (within-run) and intermediate precision (total) of the Elecsys FT4 II assay were evaluated on one cobas e 411 Immunoassay Analyzer referencing the CLSI EP5-A2 guideline. Five human serum samples and two levels of control material were analyzed. The protocol consisted of testing 2 replicates of each human serum sample and control per run, two (2) runs per day for 21 days.

Results were as follows:

| Sample | Mean (ng/dL) | Repeatability | | Intermediate Precision | | n |
| --- | --- | --- | --- | --- | --- | --- |
| | | SD (ng/dL) | CV (%) | SD (ng/dL) | CV (%) | |
| Control Level 1 | 1.22 | 0.011 | 0.9 | 0.022 | 1.8 | 84 |
| Control Level 2 | 3.11 | 0.032 | 1.0 | 0.089 | 2.9 | 84 |
| Human Serum 1 | 0.14 | 0.006 | 4.0 | 0.011 | 7.6 | 84 |
| Human Serum 2 | 1.03 | 0.013 | 1.3 | 0.023 | 2.3 | 84 |
| Human Serum 3 | 1.90 | 0.024 | 1.3 | 0.040 | 2.1 | 84 |
| Human Serum 4 | 4.93 | 0.082 | 1.7 | 0.163 | 3.3 | 84 |
| Human Serum 5 | 7.09 | 0.127 | 1.8 | 0.319 | 4.5 | 84 |

#### b. Linearity/assay reportable range:

Linearity of the Elecsys FT4 II assay was assessed on the cobas e 411 Immunoassay Analyzer referencing CLSI EP6-A.

Three high analyte serum sample pools (spiked with L-Thyroxine) were diluted with FT4 depleted human serum to create three linearity sets. For each linearity set, 13

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concentrations (11 dilutions) throughout the measuring range were prepared. All samples were assayed in triplicate within a single run. All three linearity sets generated similar results and one representative set is presented below. Linearity was evaluated using first, second and third polynomial regression analysis based on CLSI EP6-A. The coefficient of the third order polynomial regression was found to be significant; therefore, calculation of the third order model was used to demonstrate linearity from 0.095 to 8.20 ng/dL and a results summary is shown in the table below.

The linear regression line was y = 0.9752x + 0.0622

Summary of the deviation of the third order polynomial regression against the expected linear regression:

| No. of sample | Expected in ng/dL | Linear regression in ng/dL | 3^{rd} Order in ng/dL | Absolute difference in ng/dL | Relative difference in % |
| --- | --- | --- | --- | --- | --- |
| 1 | 0 | 0.052 | 0.044 | -0.008 | NA |
| 2 | 0.095 | 0.144 | 0.143 | -0.001 | -0.6 |
| 3 | 0.142 | 0.190 | 0.192 | 0.002 | 1.2 |
| 4 | 0.213 | 0.259 | 0.266 | 0.007 | 2.6 |
| 5 | 0.320 | 0.363 | 0.375 | 0.013 | 3.5 |
| 6 | 0.480 | 0.518 | 0.537 | 0.020 | 3.8 |
| 7 | 0.720 | 0.751 | 0.777 | 0.026 | 3.5 |
| 8 | 1.08 | 1.10 | 1.13 | 0.029 | 2.6 |
| 9 | 1.62 | 1.62 | 1.64 | 0.018 | 1.1 |
| 10 | 2.43 | 2.41 | 2.39 | -0.022 | -0.9 |
| 11 | 3.64 | 3.59 | 3.48 | -0.104 | -2.9 |
| 12 | 5.47 | 5.35 | 5.19 | -0.170 | -3.2 |
| 13 | 8.20 | 8.01 | 8.22 | 0.210 | 2.6 |

Based on this linearity study and the limit of detection study (below), the claimed measuring range of this assay is 0.1 – 7.7 ng/dL.

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

Traceability:

This assay has been standardized against the Elecsys FT4 method cleared under k961489. The Elecsys FT4 assay is traceable to the Enzymun-Test which was standardized using equilibrium dialysis.

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# Calibrator Stability:

When stored unopened at $2 - 8^{\circ}\mathrm{C}$ the shelf life for the calibrators is 18 months. After opening, the calibrators are stable for 12 weeks when stored at $2 - 8^{\circ}\mathrm{C}$. Onboard the cobas e 411, the calibrators are stable for up to 5 hours at $20^{\circ}\mathrm{C}$. The stability protocols and acceptance criteria were reviewed and found to be adequate.

## Value assignment for the calibrators:

The Elecsys FT4 II Calset is value assigned based on an internal value assignment procedure using the master calibrators as the reference value. The kit calibrator (CalSet) values are assigned by reading their respective concentrations from the master calibration curve. The assigned value for each level of the CalSet is the median over a minimum of 6 determinations. Kit calibrator (CalSet) values are assigned for each instrument platform. Examples of the target value for calibrator 1 and 2 are shown in the table below.

| Level | Target Value |
| --- | --- |
| Calibrator 1 | 0.78 ng/dL |
| Calibrator 2 | 3.5 ng/dL |

## d. Detection limit:

The Limit of Blank, Limit of Detection and Limit of Quantitation were evaluated referencing CLSI EP17 – A2.

The Limit of Blank (LoB) is the 95th percentile value from 60 measurements of analyte-free samples over several independent series. The LoB corresponds to the concentration below which analyte-free samples are found with a probability of $95\%$ and was determined to be $0.03\mathrm{ng / dL}$.

The Limit of Detection (LoD) is determined based on the Limit of Blank and the standard deviation of low concentration samples. The LoD corresponds to the lowest analyte concentration which can be detected (value above the LoB with a probability of $95\%$) and was determined to be $0.05\mathrm{ng / dL}$.

The Limit of Quantitation (functional sensitivity) is the lowest analyte concentration that can be reproducibly measured with an intermediate precision CV of $\leq 20\%$ and was determined to be $0.1\mathrm{ng / dL}$. Values measured as less than $0.1\mathrm{ng / dL}$ will be reported as $< 0.1\mathrm{ng / dL}$.

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The FT4 II assay has a measuring range of 0.1 to 7.77 ng/dL.

e. Analytical specificity:

1. Endogenous substances

The effect of endogenous substances was evaluated on the e 411 Analyzer using pooled human serum samples spiked with L-Thyroxine. For each potential interferent, three serum samples containing low, mid, and high concentrations of fT4 were analyzed. For all substances tested, no significant interference was defined as recovery ± 10% of initial value. The potential interferents and the highest concentration tested which did not cause significant interference are listed below:

| Potential interferent | Highest concentration at which no interference was observed |
| --- | --- |
| bilirubin | 41 mg/dL |
| hemoglobin | 1.0 g/dL |
| Lipemia (intralipid) | 2000 mg/dL |
| biotin | 20 ng/mL |
| albumin | 6.3 g/dL |
| IgG | 7 g/dL |
| IgA | 1.6 g/dL |
| IgM | 1 g/dL |
| Rheumatoid Factor | 1200 IU/mL |

2. Structurally similar substances

The effect of structurally similar substances was determined using human serum samples spiked with potential cross-reactant compounds and analyzed in duplicate on the e 411 Analyzer. For all substances tested, no significant interference was defined as recovery ± 10% of initial value. The following cross-reactivities were found at FT4 concentrations of approximately 0.974 ng/dL and 2.66 ng/dL:

| Cross-reactant | Concentration tested (ng/dL) | Cross-reactivity % |
| --- | --- | --- |
| L-T3 | 50000 | ≤ 0.005 |
| D-T3 | 50000 | ≤ 0.001 |
| rT3 | 190000 | ≤ 0.003 |
| 3-iodo-L-tyrosine | 10000000 | ≤ 0.000 |

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| Cross-reactant | Concentration tested (ng/dL) | Cross-reactivity % |
| --- | --- | --- |
| 3,5-diiodo-L-tyrosine | 10000000 | ≤ 0.000 |
| 3,3',5-triiodothyroacetic acid | 100000 | ≤ 0.0002 |
| 3,3',5,5'-tetraiodothyroacetic acid | 100000 | ≤ 0.001 |

3. Common drugs

The effect of 29 common drugs was evaluated on the e 411 Analyzer using human serum pools spiked with L-Thyroxine. For each drug, two serum samples containing a low and high concentration of FT4 were analyzed. For all substances tested, no significant interference was defined as recovery ± 10% of initial value. The drugs and the highest concentration tested which did not cause significant interference are listed below.

| Drug | Highest concentration at which no interference was observed |
| --- | --- |
| Acetaminophen | 200 μg/mL |
| Acetylcysteine | 150 μg/mL |
| Acetylsalicylic Acid | 300 μg/mL |
| Amiodarone | 200 μg/mL |
| Ampicillin-Na | 1000 μg/mL |
| Ascorbic acid | 300 μg/mL |
| Carbimazole | 6.0 μg/mL |
| Cefoxitin | 2500 μg/mL |
| Cyclosporine | 5 μg/mL |
| Doxycycline | 50 μg/mL |
| Fluocortolone | 100 μg/mL |
| Heparin | 5000 U |
| Hydrocortisone | 200 μg/mL |
| Ibuprofen | 50 μg/mL |
| Iodide | 0.2 μg/mL |
| Levodopa | 20 μg/mL |
| Methyldopa | 20 μg/mL |
| Metronidazole | 200 μg/mL |
| Octreotide | 0.3 μg/mL |
| Perchlorate | 2000 μg/mL |
| Phenylbutazone | 100 μg/mL |
| Prednisolone | 100 μg/mL |

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| Drug | Highest concentration at which no interference was observed |
| --- | --- |
| Propranolol | 240 μg/mL |
| Propylthiouracil | 300 μg/mL |
| Rifampicin | 60 μg/mL |
| Theophylline | 100 μg/mL |
| Thiamazole | 80 μg/mL |

In in vitro studies the drugs Furosemide and Levothyroxine caused elevated Free T4 findings at the daily therapeutic dosage level. In rare cases, interference due to extremely high titers of antibodies to analyte-specific antibodies, streptavidin or ruthenium can occur. Cautions regarding Furosemide, Levothyroxine, analyte - specific antibodies, streptavidin and ruthenium are stated in the labeling.

The sponsor also has the following limitation in their labeling:

"Any influence that might affect the binding behavior of the binding proteins can alter the result of the FT4 tests (e.g. drugs, NTIs (Non Thyroid Illness), patients suffering from FDH (Familial Dysalbuminemic Hyperthyroxinemia) or increased TBG in pregnancy."

"Samples should not be taken from patients receiving therapy with high biotin doses (i.e. > 5 mg/day) until at least 8 hours following the last biotin administration."

"The test cannot be used in patients receiving treatment with lipid-lowering agents containing D-T4. If the thyroid function is to be checked in such patients, the therapy should first be discontinued for 4-6 weeks to allow the physiological state to become re-established."

f. Assay cut-off:

Not applicable

2. Comparison studies:

a. Method comparison with predicate device:

A method comparison study was performed with 170 human serum samples with concentrations ranging from 0.161 to 7.05 ng/dL. Eleven samples were spiked to produce high concentrations and four samples were diluted to produce low concentrations. The comparison of the Elecsys FT4 II assay (y) with the predicate device, Elecsys FT4 assay (x), produced the following correlation:

Linear regression y = 1.02x - 0.075 r = 0.996

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b. Matrix comparison:

A matrix comparison study was performed using 72 samples collected into serum and K₂-EDTA, K₃-EDTA, and Li Heparin anticoagulant tubes. Serum concentrations ranged from 0.35 – 7.29 ng/dL. Testing was performed using the cobas e 411 Analyzer. The linear regressions results were as follows:

Serum vs. K₂-EDTA
slope 1.02
intercept -0.02
r² 0.999

Serum vs. K₃-EDTA
slope 1.00
intercept 0.00
r² 0.998

Serum vs. Lithium Heparin
slope 1.01
intercept -0.01
r² 0.998

The sponsor claims that K₂-EDTA, K₃-EDTA, and Lithium heparin plasma are acceptable anti-coagulants for the FT4 assay.

3. Clinical studies:
a. Clinical Sensitivity:
Not applicable
b. Clinical specificity:
Not applicable
c. Other clinical supportive data (when a. and b. are not applicable):
Not applicable

4. Clinical cut-off:
Not applicable

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5. Expected values/Reference range:

Euthyroid: 0.93–1.7 ng/dL

The reference range was established according to the CLSI C28-A3 guideline *Defining, Establishing and Verifying Reference Intervals in the Clinical Laboratory*; based on the capability of transferring the predicate device’s reference range to the candidate device’s reference range because of the comparability of the two testing systems. Sponsor followed the transferability guidance and verification procedure of the CLSI and has confirmed that the established reference range from the predicate device is transferable to the candidate device using 60 apparently healthy subjects (30 males and 30 females) with a normal TSH value. The reference range was previously established in k961489 for the predicate device by conducting a reference range study using 801 apparently healthy subjects.

The sponsor also states that each laboratory should investigate the transferability of the expected values to its own patient population and if necessary determine its own reference ranges in the labeling.

N. Proposed Labeling:

The labeling is sufficient and it satisfies the requirements of 21 CFR Part 809.10.

O. Conclusion:

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

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