IDS-ISYS 25-HYDROXY VITAMIN DS, AND IDS-ISYS 25 HYDROXY VITAMIN DS CONTROL SET

K140554 · Immunodiagnostic Systems , Ltd. · MRG · Dec 19, 2014 · Clinical Chemistry

Device Facts

• Record ID: K140554
• Device Name: IDS-ISYS 25-HYDROXY VITAMIN DS, AND IDS-ISYS 25 HYDROXY VITAMIN DS CONTROL SET
• Applicant: Immunodiagnostic Systems , Ltd.
• Product Code: MRG · Clinical Chemistry
• Decision Date: Dec 19, 2014
• Decision: SESE
• Submission Type: Traditional
• Regulation: 21 CFR 862.1825
• Device Class: Class 2

Intended Use

The IDS-iSYS 25-Hydroxy Vitamin DS Assay (IDS-iSYS 25OHDS) is intended for the quantitative determination of 25-hydroxy vitamin D (25OHD) and other hydroxylated metabolites in human serum on the IDS-iSYS Multi-Discipline Automated System. Results are to be used in conjunction with other clinical and laboratory data to assist the clinician in the assessment of vitamin D sufficiency in an adult population. The IDS-iSYS 25-Hydroxy Vitamin DS (25OHDS) Control Set is used for quality control of the IDS-iSYS 25-Hydroxy Vitamin DS assay on the IDS-iSYS Multi-Discipline Automated System

Device Story

Chemiluminescent immunoassay for quantitative measurement of 25-hydroxy vitamin D in human serum. Operates on IDS-iSYS Multi-Discipline Automated System. Process: sample pretreatment to denature Vitamin D Binding Protein; neutralization in buffer; addition of acridinium-labeled anti-25-OHD antibody; incubation; addition of 25-OHD-coated magnetic particles; magnetic capture; wash; trigger reagent addition. Light emission is inversely proportional to 25-OHD concentration. Used in clinical laboratories by technicians. Results assist clinicians in assessing patient vitamin D sufficiency.

Clinical Evidence

Bench testing only. Precision evaluated per CLSI EP5-A2 (n=80 per sample, CVs 7.2-10.6%). Linearity per CLSI EP-6A (R2=1.00, range 7-125ng/mL). LoB 0.6ng/mL, LoD 2.6ng/mL, LoQ 7.0ng/mL. Method comparison (n=99) against ID-LC-MS/MS RMP showed Passing-Bablok slope 0.95; comparison against predicate (n=283) showed slope 0.96. Specificity testing performed for various vitamin D metabolites and potential interferents (triglycerides, bilirubin, hemoglobin, biotin, HAMA). Reference range established in 275 healthy adults (12.7–64.2ng/mL).

Technological Characteristics

Chemiluminescent immunoassay; magnetic-particle solid phase; acridinium ester label. Reagents: MPV1 (magnetic particles), sheep polyclonal anti-25-OH D antibody, NaOH, assay buffer. Automated analyzer platform. Traceable to ID-LC-MS/MS RMP and NIST SRM 2972. Storage 2-8°C. Software-controlled automated processing.

Indications for Use

Indicated for quantitative determination of 25-hydroxyvitamin D and hydroxylated metabolites in human serum to assist clinicians in assessing vitamin D sufficiency in adult populations. Prescription use only.

Regulatory Classification

Identification

A vitamin D test system is a device intended for use in clinical laboratories for the quantitative determination of 25-hydroxyvitamin D (25-OH-D) and other hydroxylated metabolites of vitamin D in serum or plasma to be used in the assessment of vitamin D sufficiency.

Special Controls

*Classification.* Class II (special controls). Vitamin D test systems must comply with the following special controls:(1) Labeling in conformance with 21 CFR 809.10 and (2) Compliance with existing standards of the National Committee on Clinical Laboratory Standards.

Predicate Devices

• IDS-iSYS 25-Hydroxy Vitamin D (k091849)

Submission Summary (Full Text)

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

December 19, 2014

IMMUNODIAGNOSTIC SYSTEMS LTD. MICK HENDERSON, RA OFFICER 10 DIDCOT WAY, BOLDON BUSINESS PARK BOLDON, TYNE & WEAR NE35 9PD UNITED KINGDOM

Re: K140554

Trade/Device Name: IDS-iSYS 25-Hydroxy Vitamin DS Assay IDS-iSYS 25-Hydroxy Vitamin D8 Control Set Regulation Number: 21 CFR 862.1825 Regulation Name: Vitamin D test system Regulatory Class: II Product Code: MRG, JJX Dated: November 10, 2014 Received: November 10, 2014

Dear Mr. Mick Henderson:

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

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If you desire specific advice for your device on our labeling regulations (21 CFR Parts 801 and 809), please contact the Division of Industry and Consumer Education at its toll-free number [phone] or [phone] 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

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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 [phone] or [phone] or at its Internet address

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Sincerely yours,

Katherine Serrano -S

For : Courtney H. Lias, Ph.D. Director Division of Chemistry and Toxicology Devices Office of In Vitro Diagnostics and Radiological Health Center for Devices and Radiological Health

Enclosure

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DEPARTMENT OF HEALTH AND HUMAN SERVICES Food and Drug Administration

#### Indications for Use

Form Approved: OMB No.0910-0120 Expiration Date: January 31, 2017 See PRA Statement below.

510(k) Number (if known) k140554

Device Name IDS-iSYS 25-Hydroxy Vitamin DS 1DS-iSYS 25-Hydroxy Vitamin DS Control Set

Indications for Use (Describe)

The IDS iSYS 25-Hydroxy Vitamin DS Assay (IDS-iSYS 250HD*) is intended for the quantitative determination of 25-hydroxyvitamin D (25OHD) and other hydroxylated metabolites in human serum on the IDS iSYS Multi-Discipline Automated System. Results are to be used in conjunction with other clinical and laboratory data to assist the clinician in the assessment of vitamin D sufficiency in an adult population.

The IDS-iSYS 25-Hydroxy Vitamin DS (250HDS) Control Set is used for quality control of the IDSiSYS 25-Hydroxy Vitamin DS assay on the IDS-iSYS Multi-Discipline Automated System.

| Type of Use ( <i>Select one or both, as applicable</i> ) | |
|----------------------------------------------------------|-----------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------|
| | <span> <b> <svg class="bi bi-check-square-fill" fill="currentColor" height="1em" viewbox="0 0 16 16" width="1em" xmlns="http://www.w3.org/2000/svg"> <path d="M2 0a2 2 0 0 0-2 2v12a2 2 0 0 0 2 2h12a2 2 0 0 0 2-2V2a2 2 0 0 0-2-2H2zm10.03 4.97a.75.75 0 0 0-1.08.022L7.477 9.417 5.384 7.323a.75.75 0 0 0-1.06 1.06L6.97 11.03a.75.75 0 0 0 1.079-.02l3.992-4.99a.75.75 0 0 0-.01-1.05z" fill-rule="evenodd"></path> </svg> Prescription Use (Part 21 CFR 801 Subpart D) </b> </span> |
| | <span> <svg class="bi bi-square" fill="currentColor" height="1em" viewbox="0 0 16 16" width="1em" xmlns="http://www.w3.org/2000/svg"> <path d="M14 1H2a1 1 0 0 0-1 1v12a1 1 0 0 0 1 1h12a1 1 0 0 0 1-1V2a1 1 0 0 0-1-1zM2 0a2 2 0 0 0-2 2v12a2 2 0 0 0 2 2h12a2 2 0 0 0 2-2V2a2 2 0 0 0-2-2H2z" fill-rule="evenodd"></path> </svg> Over-The-Counter Use (21 CFR 801 Subpart C) </span> |

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# 510(k) SUMMARY

| 510k Number | k140554 |
|--------------|-----------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------|
| Introduction | According to the requirements of 21CFR807.92, the following<br>information provides sufficient detail to understand the basis for a<br>determination of substantial equivalence. |
| Submitter | Immunodiagnostic Systems Limited<br>10 Didcot Way<br>Boldon Business Park<br>Boldon<br>Tyne and Wear<br>NE35 9PD<br>United Kingdom<br><br>Contact Person: Mick Henderson<br>Phone: [phone]<br>Fax: [phone]<br>Email: [e-mail]<br><br>Secondary Contact: Mr. R. Prebula<br>Hogan Lovells US LLP,<br>Columbia Square,<br>555 Thirteenth Street, NW,<br>Washington DC<br>20004<br>Washington<br>Phone: [phone]<br>Fax: [phone]<br>Email: [e-mail]<br><br>Date prepared: 17, December 2014 |
| Device Name | Proprietary names: IDS-iSYS 25-Hydroxy Vitamin DS<br>IDS-iSYS 25-Hydroxy Vitamin DS Control Set |
| | Common names: As above |
| | Classification: 21CFR862.1825 Vitamin D Test System,<br>Class II<br>21CFR862.1660 Quality Control Material<br>(Assayed and Unassayed),<br>Class I, reserved |
| | Product Code: MRG |

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| Predicate Device | The IDS-iSYS 25-Hydroxy Vitamin DS is substantially equivalent to<br>other products in commercial distribution intended for similar use. We<br>claim equivalency to the currently marketed IDS-iSYS 25-Hydroxy<br>Vitamin D (k091849). |
|---------------------|----------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------|
| Device Description | The IDS-iSYS 25-Hydroxy Vitamin DS assay consists of a reagent<br>cartridge and one set of calibrators (Calibrators A & B or CAL A &<br>CAL B). The reagent cartridge contains multiple reagents: MPV1<br>(Magnetic particles coated with 25-OH D in a phosphate buffer<br>containing methanol with sodium azide as preservative), CONJ (Anti-<br>25-OH D sheep polyclonal antibody labelled with an acridinium ester<br>derivative, in buffer containing bovine, sheep, rabbit and mouse<br>proteins with sodium azide as preservative), NaOH (Sodium hydroxide<br>solution <0.5 M), and BUF (Assay buffer containing proprietary<br>displacing compounds, methanol, and sodium azide as preservative). |
| | Calibrators A and B contain horse serum in a buffer matrix with two<br>defined concentrations of 25-OH D and sodium azide as a preservative.<br>The IDS-iSYS 25-Hydroxy Vitamin DS Control Set contains horse<br>serum in a buffer matrix with three defined concentrations of 25-OH D<br>and sodium azide as a preservative. |
| Indications for Use | The IDS-iSYS 25-Hydroxy Vitamin DS assay (IDS-iSYS 25OHDS ) is<br>intended for the quantitative determination of 25-hydroxyvitamin D<br>(25OH D) and other hydroxylated metabolites in human serum on the<br>IDS-iSYS Multi-Discipline Automated System. Results are to be used<br>in conjunction with other clinical and laboratory data to assist the<br>clinician in the assessment of vitamin D sufficiency in an adult<br>population.<br>The IDS-iSYS 25-Hydroxy Vitamin DS (25OHDS) Control Set is used<br>for quality control of the IDS-iSYS 25-Hydroxy Vitamin DS assay on<br>the IDS-iSYS Multi-Discipline Automated System. |
| Conditions for use: | For in vitro diagnostic use only.<br>Rx Only: |

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## Comparison Tables

## Similarities compared to the chosen predicate device (k091849)

| Performance | IDS-iSYS 25-Hydroxy Vitamin<br>D Assay<br>Predicate device (k091849) | IDS-iSYS 25-Hydroxy Vitamin<br>Dˢ<br>Candidate device |
|-------------------------------------------|-----------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------|-------------------------------------------------------|
| Intended Use | Quantitative determination of 25-<br>Hydroxy Vitamin D and other<br>hydroxylated metabolites in<br>human serum. | Same |
| Indications for use | Results are to be used in<br>conjunction with other clinical<br>and laboratory data to assist the<br>clinician in the assessment of<br>vitamin D sufficiency in an adult<br>population. | Same |
| Analyte | 25-Hydroxy Vitamin D (25-OH<br>D) | Same |
| Calibrator matrix | Equine serum buffer matrix with<br>two defined concentrations of 25-<br>OH D and sodium azide as a<br>preservative. | Same |
| Sample matrix<br>(primary tube type) | Human Serum | Same |
| Reagent storage | 2-8 °C | Same |
| Sample preparation<br>(pre-treatment) | Performed on-board the analyzer | Same |
| Sample volume | 10μL | Same |
| Method of detection<br>(Test methodology) | Chemiluminescent immunoassay<br>using magnetic-particle solid<br>phase and acridinium label | Same |
| Automation | Fully automated assay | Same |
| Kit reagent<br>components | Reagent cartridge (1 vial each of<br>MPV1, CONJ, NaOH & BUF),<br>two concentration levels of<br>calibrators (A&B) (1 vial of each)<br>& a mini CD | Same |
| Control Kit<br>components | Three concentration levels of<br>controls (3 vials of each) & a<br>mini CD | Same |
| Calibration<br>procedure | User-initiated 2 point calibration<br>to adjust the batch related master<br>curve. The system stores the<br>calibration for the interval<br>specified in the kit IFU. | Same |
| Quality Control | Requires three buffer based<br>controls to validate the calibration | Same |

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### Kit Controls:

| Performance | IDS-iSYS 25-Hydroxy Vitamin D Control Set<br>Predicate (k091849) | IDS-iSYS 25-Hydroxy Vitamin DS Control Set |
|---------------------------|-------------------------------------------------------------------------------------------------------------|--------------------------------------------|
| Intended Use | The quality control of the assay on the IDS-iSYS. | Same |
| Control matrix | Equine serum buffer matrix with three defined concentrations of 25-OH D and sodium azide as a preservative. | Same |
| Control Kit<br>components | Three concentration levels of controls (3 vials of each) & a mini CD | Same |
| Quality Control | Requires three buffer based controls to validate the calibration | Same |
| Stability | After opening at 2 - 8 °C: To the expiry date<br>On board the analyzer: 2.5 hours | Same |
| Reagent storage | 2-8 °C | Same |

## Differences compared to the chosen (FDA cleared; marketed) predicate device

| Performance | Predicate device | Candidate device |
|-------------------------------------|------------------------------------------------------------------------------------------------|---------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------|
| Kit reagent<br>component<br>volumes | Reagent cartridge (1 vial each):<br>MPV1 (2.6mL), CONJ (7.1mL),<br>NaOH (5.2mL) & BUF (26.0mL) | Reagent cartridge (1 vial each):<br>MPV1 (2.0mL), CONJ (10.1mL),<br>NaOH (5.2mL) & BUF (26.0mL) |
| Antibodies | Anti-25 OH D Sheep Polyclonal IgG | Same, but with a different source<br>of antibody pool. |
| Traceability/<br>Standardization | Traceable to U.V. quantification. | Traceable to the isotope dilution-<br>liquid chromatography/tandem<br>mass spectrometry (ID-LC-<br>/MS/MS) 25(OH)D Reference<br>Method Procedure (RMP) which<br>was used in assigning the target<br>value for the VDSP samples.<br><br>The ID-LC-MS/MS RMP is |

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| | | traceable to the National Institute<br>of Standards and Technology<br>Standard Reference Material<br>(SRM) 2972. |
|---------------------------------------------------------|---------------------------------------------------------------|------------------------------------------------------------------------------------------------------------------|
| Calibration interval | 7 days | 14 days |
| Range of assay | 6 - 126ng/mL | 7 - 125ng/mL |
| Sensitivity | LoB: 1.8 ng/mL | LoB: 0.6 ng/mL |
| | LoD: 3.6 ng/mL | LoD: 2.6 ng/mL |
| | LoQ: 6.2 ng/mL | LoQ: 7.0 ng/mL |
| Reference range | Non-parametric reference interval: 7.9<br>- 57.8ng/mL (n=150) | Non-parametric reference<br>interval: 12.7 – 64.2ng/mL<br>(n=275) |
| Assay Duration | 38 minutes | 36 minutes |
| On board the<br>analyzer reagent<br>stability | 7 days | 21 days |
| In use (after<br>opening at 2-8°C)<br>reagent stability | 14 days | 21 days |

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

- 1. Analytical performance:
- a. Precision/Reproducibility:

A study was performed in accordance with CLSI EP5 A2 where six (6) serum samples (ranging from 12.0ng/mL to 116.5ng/mL) were assayed using three (3) lots of reagents in duplicate (n=2) twice per day for 20 days on three (3) analyzers.

Representative LOT

| Sample | n | mean | Within-run | | Total | |
|---------|----|---------|------------|------|-------|-------|
| | | (ng/mL) | SD | CV% | SD | CV% |
| Serum 1 | 80 | 13.2 | 0.8 | 6.4% | 1.4 | 10.6% |
| Serum 2 | 80 | 27.2 | 1.5 | 5.4% | 2.5 | 9.0% |
| Serum 3 | 80 | 38.9 | 2.3 | 5.8% | 3.5 | 9.1% |
| Serum 4 | 80 | 54.5 | 3.2 | 5.8% | 5.0 | 9.1% |
| Serum 5 | 80 | 77.2 | 4.0 | 5.2% | 6.6 | 8.5% |
| Serum 6 | 80 | 119.9 | 6.1 | 5.1% | 8.6 | 7.2% |

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

Linearity was evaluated based on CLSI EP-6A, "Evaluation of the Linearity of Quantitative Measurement Procedures: A Statistical Approach". In the linearity study, samples were prepared by diluting a high serum sample with a low serum sample to obtain eleven (11) concentration levels across the measuring range. One study was performed with one manufacturing batch resulting in a total of 22 concentration levels ranging from 5.8ng/mL to 152.9ng/mL. Each concentration level was assayed in duplicate, and the resulting mean concentrations were compared to predicted concentrations.

The resulting linear regression equation was y = 0.96x + 2.1ng/mL, R2 = 1.00

The reportable range of the assay is 7-125ng/mL.

- c. Traceability, Stability, Expected values (controls, calibrators, or methods):
#### Calibrator traceability and value assignment

An internal stock solution is prepared by reconstituting a vial of 25hydroxyvitamin D3 with ethanol and adding this to horse serum. The potency assigned to the stock solution is nominally based on the absorbance at 264nm of the ethanolic 25D solution prior to addition to the serum. A molar extinction coefficient of 18,200 is used to calculate the concentration from the absorbance value. This stock solution is used to prepare master calibrators, whose final value assignment is based on results from running the calibrators in multiple assays on multiple analyzers.

Kit calibrators are lot specific for each assay kit. Master calibrators and kit calibrators A and B are prepared gravimetrically from the stock solution or an intermediate stock solution. For value assignment, the kit calibrators are tested as unknowns in a minimum of 20 assay runs using one analyzer. Each run is calibrated using master calibrators. The values are then adjusted to ensure batch-to-batch consistency where necessary, and to facilitate the alignment to the CDC VDSP program, based on multiple correlation assays of an established patient library panel. The final assigned values obtained for the kit calibrators are verified on three additional analyzers. The values must fall within specified acceptable ranges.

The assay is traceable to the isotope dilution-liguid chromatography/tandem mass spectrometry (ID-LC-/MS/MS) 25(OH)D Reference Method Procedure (RMP) which was used in assigning the target value for the VDSP samples.

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The ID-LC-MS/MS RMP is traceable to the National Institute of Standards and Technology Standard Reference Material (SRM) 2972.

#### Stability

As there has been no change to the kit calibrators or kit controls formulation, design or intended use, the cleared IDS-iSYS 25-Hydroxy Vitamin D product (k091849) should be referred to for the relevant performance and stability details. The calibrator and control shelf-life and open-vial stability testing protocols and acceptance criteria were described and found to be adequate. Once opened calibrators and controls are stable for up to 2.5 hours on board the analyzer. Current accelerated shelf life studies support the assigned sixmonth shelf life, with real-time studies on-going.

#### d. Detection limit:

The Limit of Blank (LoB), Limit of Detection (LoD) and Limit of Quantification (LoQ) studies were performed according to CLSI EP-17A.

To establish the Limit of Blank (LoB), the zero calibrator was assayed as 10 replicates over 10 assays on 10 separate days giving a total of 100 measurements. The Limit of Blank was determined by calculating the concentration corresponding to the mean zero calibrator minus two standard deviations from the 4PL calibration curve. The LoB claim is 0.6ng/mL.

The Limit of Detection (LoD) study was performed by assaying 10 samples (native and/or diluted) with very low vitamin D concentrations (ranging from 1.9ng/mL to 8.0ng/mL) in duplicate over 12 assays spanning multiple days giving a total of 240 data points. The LoD claim is 2.6ng/mL.

The Limit of Quantification (LoQ) was determined by measuring 13 low samples (native and /or diluted) in a concentration range of 2.8 - 24.6ng/mL in duplicate over 7 individual assays spanning multiple days giving 182 data points. The LoQ claim is 7.0ng/mL and was calculated by interpolating the concentration of the analyte from the regression curve at 20% precision C.V.

#### e. Analytical specificity:

Cross-reactivity by adding endogenous 25(OH) vitamin D metabolite to serum samples: Endogenous 25(OH) vitamin D metabolites were spiked into vitamin D serum samples and analyzed with the IDS-iSYS 25-Hydroxy Vitamin Do assay. The value of un-spiked sample and spike concentration were determined by LC-MS/MS 25(OH)D method. The spiked sample was measured with the IDS-iSYS 25-Hydroxy Vitamin De assay. The % cross-reactivity was calculated based on following equation:

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## Mean conc. of spiked sample – mean conc. of unspiked sample_ x100% Spiked concentration

| Cross<br>Reactant | Spike Conc.<br>(ng/mL) | Un-spiked<br>sample<br>value<br>(ng/mL) | Spiked<br>sample<br>value<br>(ng/mL) | % Cross<br>Reactivity | Mean %<br>Cross<br>Reactivity |
|-------------------|------------------------|-----------------------------------------|--------------------------------------|-----------------------|-------------------------------|
| 25(OH)D3 | 10.0 | 20.2 | 29.2 | 90% | 97% |
| | 20.0 | 14.3 | 35.1 | 104% | |
| 25(OH)D2 | 10.0 | 16.1 | 28.5 | 124% | 120% |
| | 20.0 | 11.1 | 34.4 | 117% | |
| 24,25(OH)2D3 | 5.0 | 66.2 | 72.4 | 124% | 124% |

- b. Cross-reactivity by adding exogenous synthetic 25(OH) vitamin D metabolite to serum samples: Exogenous synthetic 25(OH) vitamin D metabolites were spiked into vitamin D serum samples. The un-spiked and spiked samples were measured with the IDS-iSYS 25- Hydroxy Vitamin Ds assay. The % cross reactivity was calculated based on the following equation:
Mean conc. of spiked sample - mean conc. of unspiked sample _ x100% Spiked concentration

| Cross Reactant | Spiked<br>conc.<br>(ng/mL) | Sample<br>Native<br>conc.<br>(ng/mL) | % Cross<br>reactivity | Mean %<br>Cross<br>reactivity |
|---------------------------------|----------------------------|--------------------------------------|-----------------------|-------------------------------|
| 3-epi-25(OH)D3 | 100.0 | 9.4 | 0% | |
| | 100.0 | 36.4 | 3% | 1% |
| 3-epi-25(OH)D2 | 100.0 | 8.6 | 0% | |
| | 100.0 | 33.4 | 2% | 1% |
| 1,25-(OH)2 D3 | 2.0 | 7.1 | 19% | |
| | 2.0 | 31.2 | -65% | -23% |
| 1,25-(OH)2 D2 | 20.0 | 8.6 | 13% | |
| | 20.0 | 37.9 | 5% | 9% |
| Vitamin D3<br>(Cholecalciferol) | 1000.0 | 6.9 | 0% | |
| (Cholecalciferol) | 1000.0 | 30.8 | 0% | 0% |
| Vitamin D2<br>(Ergocalciferol) | 100.0 | 7.4 | 1% | |
| (Ergocalciferol) | 100.0 | 37.3 | -2% | 0% |
| Paricalcitol | 100.0 | 8.2 | 0% | |
| | 100.0 | 33.4 | 2.9% | 0% |

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The following potentially interfering endogenous substances listed within the package insert do not interfere in the IDS-iSYS 25-Hydroxy Vitamin De assay when the concentrations presented in the following table are below the stated threshold, using a criterion of ±10% bias of the control sample to the test sample. Rheumatoid Factor was assessed using a recovery study with two 'normal' samples; the specification is based on recovery criteria of 90 - 110%. RF does not show significant interference up to 1500 IU/L tested. To evaluate potential lipid interference a natural specimen with 500mg/dL lipid concentration was obtained. A portion was delipidated and assayed along with the same unaltered specimen n=23 in the IDS-iSYS 25-Hydroxy Vitamin De assay.

| Potentially<br>Interfering Agent | Threshold<br>Concentration | Sample 25OHD<br>Concentration | n | Bias (Test vs.<br>Control<br>Sample) |
|----------------------------------|----------------------------|-------------------------------|---------|--------------------------------------|
| Triglycerides | 500mg/dL | 50.0ng/mL & 87.9ng/mL | 26 & 26 | -6% & -5% |
| Bilirubin,<br>conjugated | 30mg/dL | 18.0ng/mL & 65.7ng/mL | 26 & 26 | 9.2% & 6.4% |
| Haemoglobin | 40mg/dL | 19.2ng/mL & 71.7ng/mL | 26 & 26 | -8% & -2% |
| Biotin | 300nmol/L | 17.0ng/mL & 66.2ng/mL | 26 & 26 | 1% & -3% |
| HAMA | 500ng/mL | 18.2ng/mL & 72.8ng/mL | 25 & 26 | -5% & -2% |
| Red Blood Cells | 0.2% | 15.5ng/mL & 65.8ng/mL | 26 & 26 | -10% & -2% |
| Vitamin DBP | 2000ng/mL | 18.9ng/mL & 76.0ng/mL | 24 & 26 | 0% & 0% |

The package insert also states that the presence of haemoglobin at concentrations >40mg/dL might lead to falsely depressed values. Do not use hemolyzed samples.

- Assay cut-off: f.
Not applicable

### 2. Comparison studies:

### a. Method comparison with predicate device:

A method comparison study was performed to demonstrate the accuracy of the newly aligned assay. With the aligned assay calibration parameters, 99 samples in the range of 9.0ng/mL to 98.6ng/mL, by ID-LC-MS/MS RMP, were used to assess the IDSiSYS 25-Hydroxy Vitamin D assay traceability against the ID-LC-MS/MS RMP.

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The relationship between the IDS-iSYS (y) and the ID-LC-MS/MS RMP (x) is described using Passing-Bablok regression & Deming regression:

Passing Bablok regression: IDS-iSYS = 0.95 x (ID-LC-MS/MS RMP)+ 0.80ng/mL 95 % CI of the slope: 0.86 to 1.04 95 % CI of the intercept: -1.32 to 3.08ng/mL

Deming regression: IDS-iSYS = 0.94 x (ID-LC-MS/MS RMP)+1.34ng/mL 95 % CI of the slope: 0.86 to 1.01 95 % CI of the intercept: -0.78 to 3.45ng/mL

Pearson correlation coefficient, r: 0.925

In addition, the following method comparison study was performed to characterize the comparison of the newly aligned IDS-iSYS 25-Hydroxy Vitamin De assay to the unaligned IDS-iSYS 25-Hydroxy Vitamin D assay:

Method comparison studies were performed following CLSI EP9-A2. An internal method comparison study was performed with a total of 283 European-sourced serum samples tested in singlicate on the IDS-iSYS 25-Hydroxy Vitamin D Assay and the predicate (the IDS-iSYS 25-Hydroxy Vitamin D Assay). Samples spanned the assay range with values from 7.3 -115.1 ng/mL (candidate values).

Passing-Bablok regression analysis was performed to produce a summary of results for each study, as shown below.

| Passing Bablok | y = 0.96x + 1.1ng/mL |
|------------------------------------|----------------------|
| Slope, 95% Confidence Interval | 0.91 to 1.01 |
| Intercept, 95% Confidence Interval | -0.3 to 2.3ng/mL |
| Correlation Coefficient, r | 0.94 |
| n | 283 |
| Range | 7.3 to 115.1 ng/mL |

Internal Study:

- a. Matrix comparison:
Not applicable. Only serum may be used.

- 3. Expected values/Reference range:

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

An expected values study performed according to the non-parametric method in CLSI protocol C28-A2.

Samples from 275 apparently healthy light skin and dark skin male and female adults living in the United States, aged 21 to 77 years, in geographical diverse regions of the United States to represent a broad spectrum of UV light exposure in the intended population were assayed in the IDS-iSYS 25-Hydroxy Vitamin DS Assay.

The 95% reference interval was calculated by a non-parametric method following C28-A2. The following range was obtained:

Normal Adults 12.7 - 64.2ng/mL (n=275)

| | n | Observed Sample Range | Median |
|------------------|-----|-----------------------|-----------|
| Non-supplemented | 220 | 11.9 - 91.9ng/mL | 31.8ng/mL |
| Northern US | 116 | 11.9 - 55.4ng/mL | 29.7ng/mL |
| Southern US | 104 | 14.3 - 91.9ng/mL | 36.3ng/mL |
| Supplemented | 55 | 8.2 - 83.8ng/mL | 35.3ng/mL |
| Overall | 275 | 8.2 - 91.9ng/mL | 32.5ng/mL |

Observed sample ranges were:

The package insert states that there is no universal agreement on the optimal concentration of 250HD. Ranges should be based on clinical decision values that apply to both sexes of all ages rather than population based reference ranges for 25OHD.

### Conclusion:

The IDS-iSYS 25-Hydroxy Vitamin DS and IDS-iSYS 25-Hydroxy Vitamin DS Control Set data presented and provided is complete and supports the basis for substantial equivalence to the predicate device.