IDS-iSYS 25VitDs, IDS-iSYS 25VitDs Control Set

K161831 · Immunodiagnostic Systems , Ltd. · MRG · Nov 15, 2016 · Clinical Chemistry

Device Facts

• Record ID: K161831
• Device Name: IDS-iSYS 25VitDs, IDS-iSYS 25VitDs Control Set
• Applicant: Immunodiagnostic Systems , Ltd.
• Product Code: MRG · Clinical Chemistry
• Decision Date: Nov 15, 2016
• Decision: SESE
• Submission Type: Traditional
• Regulation: 21 CFR 862.1825
• Device Class: Class 2

Indications for Use

The IDS-iSYS 25 VitD⁵ Assay is intended for the quantitative determination of total 25-hydroxyvitamin D [(25(OH)D] in human serum or plasma 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 VitD⁵ Control Set is used for quality control of the IDS-iSYS 25 VitD⁵ assay on the IDS-iSYS Multi-Discipline Automated System.

Device Story

The IDS-iSYS 25 VitD assay is a chemiluminescent immunoassay performed on the IDS-iSYS Multi-Discipline Automated System. It uses magnetic particles coated with streptavidin, an acridinium ester-labeled 25(OH)D tracer, and a biotin-labeled anti-25(OH)D sheep polyclonal antibody. The system performs automated sample pre-treatment and detection. The device is used in clinical laboratories by trained personnel. The output is a quantitative concentration of 25(OH)D (ng/mL or nmol/L), which clinicians use alongside other laboratory and clinical data to evaluate a patient's vitamin D status. The assay benefits patients by providing standardized, automated measurement of vitamin D levels to guide clinical management of sufficiency.

Clinical Evidence

Bench testing only. Precision study (n=80-244 per sample) showed total CVs 4.9-9.4%. Linearity confirmed 4-110 ng/mL (R^2=1.00). Method comparison against ID-LC-MS/MS reference method (n=136) showed slope 0.99, intercept -0.51, r=0.97. Interference testing performed for endogenous substances (e.g., biotin, bilirubin, HAMA, lipids). Reference interval study (n=392) established range 10.4-59.5 ng/mL.

Technological Characteristics

Chemiluminescent immunoassay; magnetic-particle solid phase; acridinium label. Reagents: streptavidin-coated magnetic particles, anti-25(OH)D sheep polyclonal antibody, acridinium-labeled 25(OH)D. Fully automated on IDS-iSYS system. Traceable to ID-LC-MS/MS reference method and NIST SRM 2972. Storage 2-8°C. Sample types: serum, K2 EDTA, lithium heparin, sodium heparin.

Indications for Use

Indicated for the quantitative determination of total 25-hydroxyvitamin D in human serum or plasma to assist clinicians in assessing vitamin D sufficiency in adult populations.

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^S Assay (k140554)
• IDS-iSYS 25-Hydroxy Vitamin D^S Control Set (k140554)

Submission Summary (Full Text)

{0}

1

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

A. 510(k) Number:
k161831

B. Purpose for Submission:
New device

C. Measurand:
Total 25-hydroxyvitamin D

D. Type of Test:
Quantitative chemiluminescent immunoassay

E. Applicant:
Immunodiagnostic Systems Limited

F. Proprietary and Established Names:
IDS-iSYS 25VitD
IDS-iSYS 25VitD Control Set

G. Regulatory Information:
1. Regulation section:
21 CFR 862.1825, Vitamin D Test System
21 CFR 862.1660, Quality Control Material
2. Classification:
Class II
Class I, reserved
3. Product code:

{1}

MRG, Vitamin D Test System

JJX, Single (specified) Analyte Controls (Assayed and Unassayed)

4. Panel:
Clinical Chemistry (75)

H. Intended Use:

1. Intended use(s):
See indications for use below.

2. Indication(s) for use:
The IDS-iSYS 25 VitD⁵ Assay is intended for the quantitative determination of total 25-hydroxyvitamin D [(25(OH)D] in human serum or plasma 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 VitD⁵ Control Set is used for quality control of the IDS-iSYS 25 VitD⁵ assay on the IDS-iSYS Multi-Discipline Automated System.

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

4. Special instrument requirements:
For use on the IDS-iSYS Multi-Discipline Automated System.

I. Device Description:
The IDS-iSYS 25VitD⁵ assay consists of a reagent cartridge (with multiple components) and one set of calibrators.

Reagent Cartridge:
MPV1 – Magnetic particles coated with streptavidin in phosphate buffer with sodium azide (<0.1%) as preservative (1 bottle, 2.5 mL)
NaOH – Sodium hydroxide solution (<0.5 M) (1 bottle, 13.0 mL)
25D ACR – 25D labelled with an acridinium ester derivative, in buffer containing bovine serum albumin with sodium azide (<0.1%) as preservative (1 bottle, 9.0 mL)

2

{2}

Ab-BIOT - Anti-25(OH)D sheep polyclonal antibody labelled with an biotin, in buffer containing bovine, sheep and mouse proteins with sodium azide (<0.1 %) as preservative (1 bottle, 9.0 mL)

BUF - Assay buffer containing proprietary displacing compounds, methanol (>10 % but <20%) and sodium azide (<0.1 %) as preservative (1 bottle, 23.0 mL)

Calibrators:

Kit Calibrators A and B (CAL A & CAL B) (1 bottle of each, 1.5 mL per bottle) contain human serum buffer matrix with two defined concentrations of 25(OH)D and sodium azide (<0.1%) as preservative.

Control Set:

Controls 1 and 2 (CTL 1 & CTL 2) (3 bottles of each, 2.5 mL per bottle) contain human serum buffer matrix with two defined concentrations of 25(OH)D and sodium azide (<0.1%) as preservative. The target concentrations are 17 ng/mL and 90 ng/mL.

Statement in labeling regarding the use of human blood-based materials in calibrators and controls:

Human material used in the preparation of this product has been tested by FDA recommended assays for the presence of antibody to Human Immunodeficiency Virus (HIV I and II), Hepatitis B surface antigen, antibody to Hepatitis C, and found negative.

J. Substantial Equivalence Information:

1. Predicate device name(s):

IDS-iSYS 25-Hydroxy Vitamin D$^{\text{S}}$ Assay

IDS-iSYS 25-Hydroxy Vitamin D$^{\text{S}}$ Control Set

2. Predicate 510(k) number(s):

k140554

3. Comparison with predicate:

| Assay: Similarities | | |
| --- | --- | --- |
| Item | Candidate Device
IDS-iSYS 25VitD$^{\text{S}}$ | Predicate
IDS-iSYS 25-Hydroxy
Vitamin D$^{\text{S}}$
(k140554) |
| Intended Use | Same | For the quantitative
determination of 25-
hydroxyvitamin D in human
blood samples. |
| Analyte | Same | 25-Hydroxy Vitamin D
(25(OH)D) |

{3}

| Assay: Similarities | | |
| --- | --- | --- |
| Item | Candidate Device IDS-iSYS 25VitDS | Predicate IDS-iSYS 25-Hydroxy Vitamin D$ (k140554) |
| Reagent Storage | Same | 2-8 °C |
| Sample preparation (pre-treatment) | Same | Performed on-board the analyzer |
| Sample volume | Same | 10μL |
| Method of detection (Test methodology) | Same | Chemiluminescent immunoassay using magnetic-particle solid phase and acridinium label |
| Automation | Same | Fully automated assay |
| Calibration procedure | Same | User-initiated 2 point calibration to adjust the batch related master curve. The system stores the calibration for the interval specified in the kit IFU. |
| Traceability/Standardization | Same | Traceable to the isotope dilution-liquid 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. The ID-LC-MS/MS RMP is traceable to the National Institute of Standards and Technology Standard Reference Material (SRM) 2972. |
| On board the analyzer reagent stability | Same | 21 days |

{4}

| Assay: Differences | | |
| --- | --- | --- |
| Item | Candidate Device IDS-iSYS 25VitDS | Predicate IDS-iSYS 25-Hydroxy Vitamin D$ (k140554) |
| Kit Calibrator matrix | Human serum buffer matrix with two defined concentrations of 25(OH)D and sodium azide as a preservative. | Equine serum buffer matrix with two defined concentrations of 25(OH)D and sodium azide as a preservative. |
| Kit reagent components | Reagent cartridge (1 vial each of MPV1, NaOH, 25D-ACR, Ab-BIOT & BUF), two concentration levels of calibrators (A&B) (1 vial of each) & a mini CD | Reagent cartridge (1 vial each of MPV1, CONJ, NaOH & BUF), two concentration levels of calibrators (A&B) (1 vial of each) & a mini CD |
| Control Kit components | Two concentration levels of controls (3 vials of each) | Three concentration levels of controls (3 vials of each) |
| Kit reagent component volumes | Reagent cartridge (1 vial each): MPV1 (2.5mL), NaOH (13.0mL), 25D-ACR (9.0mL), Ab-BIOT (9.0mL) & BUF (23.0mL) | Reagent cartridge (1 vial each): MPV1 (2.0mL), CONJ (10.1mL), NaOH (13mL) & BUF (26.0mL) |
| Antibodies | Same, but with a different source of antibody pool | Anti-25 OH D Sheep Polyclonal IgG |
| Calibration interval | 7 days | 14 days |
| Range of assay | 4.00 – 110 ng/mL | 7 – 125 ng/mL |
| Sensitivity | LoB: 1.31 ng/mLLoD: 1.98 ng/mLLoQ: 3.53 ng/mL | LoB: 0.6 ng/mLLoD: 2.6 ng/mLLoQ: 7.0 ng/mL |
| Expected values | 10.4 to 59.5 ng/mL | 12.7 to 64.2 ng/mL |
| In use (after opening at 2-8°C) reagent stability | 42 days | 21 days |
| Sample type | Serum (standard sampling tubes or tubes containing serum separating gel) or plasma (K2 EDTA, lithium heparin, sodium heparin) | Serum |

{5}

| Controls: Similarities | | |
| --- | --- | --- |
| Item | Candidate Device IDS-iSYS 25VitDS | Predicate IDS-iSYS 25-Hydroxy Vitamin D$ (k140554) |
| Intended Use | Same | The quality control of the 25-OH vitamin D assay on the IDS-iSYS. |
| Stability | Same | After opening at 2 - 8 °C: To the expiry date |
| Reagent storage | Same | 2-8 °C |
| Controls: Differences | | |
| --- | --- | --- |
| Item | Candidate Device IDS-iSYS 25VitDS | Predicate IDS-iSYS 25-Hydroxy Vitamin D$ (k140554) |
| Control matrix | Human serum buffer matrix with two defined concentrations of 25-OH D and sodium azide as a preservative. | Equine serum buffer matrix with three defined concentrations of 25-OH D and sodium azide as a preservative. |
| Stability | On board the analyzer: 4 hours | On board the analyzer: 2.5 hours |
| Control levels | Level 1: 17 ng/mL
Level 2: 90 ng/mL | Level 1: 12.0-18.0 ng/mL
Level 2: 26.4-39.6 ng/mL
Level 3: 59.0-86.0 ng/mL |

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

- CLSI EP05-A2 Evaluation of Precision performance of Quantitative Measurement Methods; Approved Guideline.
- CLSI EP06-A, Evaluation of the Linearity of Quantitative Measurement Procedures: A Statistical Approach; Approved Guideline.
- CLSI EP07-A2, Interference Testing in Clinical Chemistry; Approved Guideline.
- CLSI EP09-A3, Measurement Procedure Comparison and Bias Estimation Using Patient Samples; Approved Guideline.
- CLSI EP17-A, Protocols for Determination of Limits of Detection and Limits of Quantitation; Approved Guideline.
- CLSI C28-A3, Defining, Establishing, and Verifying Reference Intervals in the Clinical Laboratory; Approved Guideline.

{6}

L. Test Principle:

The assay is based on chemiluminescence technology. 10 µL of patient sample is subjected to a pre-treatment step to denature the vitamin D binding protein (VDBP). The treated samples are then neutralised in assay buffer and a specific anti-25(OH)D antibody labelled with biotin is added. Following an incubation step, acridinium labelled 25(OH)D is added. Following a further incubation step, the magnetic particles linked to streptavidin are added. After the final incubation step the complex is captured using a magnet and a wash step performed to remove any unbound analyte. Trigger reagents are added and the resulting light emitted by the acridinium label is inversely proportional to the concentration of 25(OH)D in the original sample.

M. Performance Characteristics (if/when applicable):

1. Analytical performance:

a. Precision/Reproducibility:

A precision study was performed in accordance with CLSI EP5-A2. Nine serum samples (all native except Serum 9 that was spiked to achieve a high concentration) and two controls were assayed using three lots of reagents in duplicate, twice per day for 20 days on three to four analyzers. All three lots generated similar precision results. The within-run and total precision results are summarized in the table below using one representative lot of reagents:

| | | | Within Run | | Total | |
| --- | --- | --- | --- | --- | --- | --- |
| | n | Mean Conc. ng/mL | SD | %CV | SD | %CV |
| Serum 1 | 80 | 13.9 | 0.7 | 5.0 | 1.0 | 7.4 |
| Serum 2 | 80 | 16.9 | 0.7 | 3.9 | 1.2 | 7.2 |
| Serum 3 | 80 | 28.6 | 1.1 | 3.9 | 1.9 | 6.5 |
| Serum 4 | 80 | 37.0 | 1.3 | 3.5 | 1.8 | 4.9 |
| Serum 5 | 80 | 49.8 | 2.0 | 4.0 | 2.9 | 5.8 |
| Serum 6 | 80 | 61.1 | 2.4 | 3.9 | 3.7 | 6.0 |
| Serum 7 | 80 | 62.1 | 2.8 | 4.4 | 4.0 | 6.4 |
| Serum 8 | 80 | 93.0 | 3.5 | 3.8 | 5.8 | 6.2 |
| Serum 9 | 80 | 103.0 | 4.8 | 4.7 | 6.3 | 6.1 |

{7}

The within-run and total precision results for the combined lots of reagents are shown in the table below:

| | | | Within Run | | Total | |
| --- | --- | --- | --- | --- | --- | --- |
| | n | Mean Conc. ng/mL | SD | %CV | SD | %CV |
| Serum 1 | 244 | 14.1 | 0.7 | 5.2 | 1.3 | 9.4 |
| Serum 2 | 244 | 17.2 | 0.7 | 4.2 | 1.5 | 8.8 |
| Serum 3 | 244 | 29.1 | 1.2 | 4.0 | 2.2 | 7.4 |
| Serum 4 | 244 | 37.6 | 1.2 | 3.2 | 2.6 | 6.8 |
| Serum 5 | 244 | 50.8 | 1.7 | 3.3 | 3.4 | 6.6 |
| Serum 6 | 244 | 61.0 | 2.3 | 3.7 | 4.0 | 6.6 |
| Serum 7 | 244 | 62.9 | 2.2 | 3.4 | 4.1 | 6.6 |
| Serum 8 | 244 | 93.4 | 3.1 | 3.3 | 6.0 | 6.4 |
| Serum 9 | 244 | 102.0 | 5.0 | 4.9 | 8.7 | 8.6 |

# b. Linearity/assay reportable range:

Linearity was evaluated based on CLSI EP6-A. A high human serum sample (pool of two endogenous spiked serum samples), a low human serum sample (low serum sample diluted in zero matrix), and nine evenly spaced dilutions (created by mixing the high and low sample) were analyzed in replicates of four with sample range tested between 3.6 to $135.9\mathrm{ng / mL}$ . The concentrations tested were as follows: 3.6, 16.8, 30.0, 43.3, 56.5, 69.8, 83.0, 96.2, 109.5, 122.7, and $135.9\mathrm{ng / mL}$ . Linearity curves of the observed concentration versus the expected concentration were plotted using unweighted linear regression.

The resulting linear regression equation was $y = 1.00x + 3.75 \, \mathrm{ng/mL}$ , $R^2 = 1.00$

The results of the linearity study support the sponsors claim that the assay is linear in the assay's reportable range of 4 to $110\mathrm{ng / mL}$ .

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

# Standardization and Traceability

The IDS-iSYS 25 $\mathrm{VitD}^{\mathrm{S}}$ assay is traceable to the Ghent University isotope dilution-liquid chromatography/tandem mass spectrometry (ID-LCMS/MS) 25(OH)D Reference Method Procedure (RMP). The ID-LCMS/MS RMP is further traceable to the National Institute of Standards and Technology Standard Reference Material (NIST SRM) 2972.

This assay has been standardized in accordance with the Vitamin D Standardization Program (VDSP). Please refer to https://ods.od.nih.gov/Research/vdsp.aspx for more information on the VDSP program. To achieve standardization, the IDS-iSYS 25 $\mathrm{VitD}^{\mathrm{S}}$ assay master calibration parameters were aligned to the VDSP by using 136 single-donor human serum samples from the VSDP program, which were value assigned using the ID-LCMS/MS RMP.

{8}

9

# Calibrator and control value assignment

The IDS-iSYS 25VitD® kit calibrators are value assigned using the internal reference calibrators through an internal procedure. The IDS-iSYS 25VitD® internal reference calibrators are value assigned by assignment to samples with known levels of 25(OH)D as determined by the ID-LC-MS/MS reference method procedure. The kit calibrators are tested as unknowns in a minimum of 20 assay runs on one iSYS instrument. Each run uses secondary standards (IRs), and the concentrations of the Kit Calibrators are calculated from these secondary standards (IRs). Following assignment, the kit calibrator values are then verified over 3 assays using the full curve parameters in addition to the established calibrator values. The values must fall within specified acceptable ranges. The kit calibrator concentrations are reagent batch specific and linked together. The calibrator nominal ranges are listed below:

Calibrator A Nominal Range: 8.0 – 12.0 ng/mL
Calibrator B Nominal Range: 80.0 – 90.0 ng/mL

For kit control value assignment, the kit controls are tested as unknowns in a minimum of 21 assay runs using multiple systems. The established values are then verified in a single assay in an approved kit combination. The values must fall within specified acceptable ranges. The control nominal ranges are listed below:

Kit Control 1 Nominal Range: 12.0 – 18.0 ng/mL
Kit Control 2 Nominal Range: 65.0 – 85.0 ng/mL

# Stability

The calibrator and control shelf-life and open-vial stability testing protocols and acceptance criteria were reviewed and found to be adequate. The open-kit stability study demonstrates that the calibrators and controls are stable for 42 days once opened and stored at 2 – 8°C in an upright position in the dark. Calibrators and controls are stable for up to 4 hours on board the analyzer. Real-time stability studies are on-going to support a kit calibrator and control minimum shelf life of 12 months.

## d. Detection limit:

The limit of blank (LoB), limit of detection (LoD) and limit of quantitation (LoQ) were determined with guidance from CLSI EP17-A.

To establish the Limit of Blank (LoB), the zero calibrator was assayed in duplicate on three analyzers, with three reagent lots over multiple days for a total of 6 assays and 60 replicates per reagent lot. LoB was determined by the following equation:

$$
\mathrm{LoB} = \mathrm{mean}(\mathrm{blank}) + [1.645 \times \mathrm{SD}(\mathrm{blank})]
$$

The LoB claim is 1.31 ng/mL.

The Limit of Detection (LoD) study was performed by assaying six serum samples with very low vitamin D concentrations in duplicate on three analyzers with three

{9}

reagent lots over multiple days for a total of 6 assays and 72 replicates per reagent lot. LoD was calculated using the following equation:

$$
\mathrm {L o D} = \mathrm {L o B} + 1. 6 4 5 \mathrm {x} \text {p o o l e d S D}
$$

The LoD claim is $1.98\mathrm{ng / mL}$

The Limit of Quantification (LoQ) was determined by measuring ten serum samples with low vitamin D concentrations (ranging from 0.994 to $9.32\mathrm{ng / mL}$ ) in duplicate on three analyzers with three reagent lots over multiple days for a total 120 replicates per reagent lot. The LoQ claim is $3.53\mathrm{ng / mL}$ and is defined as concentration interpolated from the regression curve where the upper $95\%$ confidence interval for the curve has a $20\%$ CV.

The LoB, LoD and LoQ are summarized below:

| LoB | LoD | LoQ |
| --- | --- | --- |
| 1.31 ng/mL | 1.98 ng/mL | 3.53 ng/mL |

The reportable range of the assay is 4 to $110\mathrm{ng / mL}$

# e. Analytical specificity:

# Interference

Interference testing was performed based on CLSI EP7-A2 to assess common or known substances that could interfere with the IDS-iSYS $25\mathrm{VitD}^{\mathrm{S}}$ assay. The potential endogenous interferents listed below were spiked into two human serum samples that contained two different concentrations of 25(OH)D. Each sample was tested in replicates of 26. The 25(OH)D values of the spiked samples were compared to the control samples containing no interferent. Significant interference was defined as greater than $10\%$ difference from the expected concentration. The interference study results are summarized in the following table:

| Potential endogenous interferent | 25(OH)D concentration of non-spiked sample (ng/mL) | Highest Test Concentration that demonstrated no significant interference |
| --- | --- | --- |
| Triglycerides (Intralipid) | 31.4 and 61.4 | 500 mg/dL |
| Hemoglobin | 33.2 and 54.8 | 500 mg/dL |
| Bilirubin, conjugated | 30.3 and 59.4 | 20 mg/dL |
| Bilirubin, unconjugated | 23.2 and 67.4 | 20 mg/dL |
| Total Protein | 32.0 and 30.5 | 10 g/dL |
| Human Anti Mouse Antibody (HAMA) | 33.8 and 65.6 | 1000 ng/mL |
| Red Blood Cells | 29.8 and 57.7 | 0.2% |
| Vitamin D Binding Protein | 30.0 and 61.7 | 2000 ng/mL |

{10}

| Potential endogenous interferent | 25(OH)D concentration of non-spiked sample (ng/mL) | Highest Test Concentration that demonstrated no significant interference |
| --- | --- | --- |
| Biotin | 34.9 and 64.6 | 200 nM |
| Acetaminophen | 34.7 and 60.2 | 200 μg/mL |
| Ibuprofen | 32.6 and 66.7 | 140 μg/mL |
| Carbamazepine | 28.3 and 67.4 | 30 μg/mL |
| Phenytoin | 32.2 and 62.2 | 50 μg/mL |

Rheumatoid Factor (Rf): Interference from Rf was assessed by spiking different amounts of a high Rf sample into two serum base samples containing two different concentrations of 25(OH)D. Each sample was tested in replicates of 4. The concentration of 25(OH)D in the Rf spike was determined in the high Rf sample (diluted to a level where Rf interference would not be expected to occur prior to 25(OH)D determination). The observed 25(OH)D values of the spiked samples were compared to the expected 25(OH)D values contributed by the base samples and high Rf sample. Significant interference was defined as greater than 10% difference from the expected concentration. The results are summarized in the following table:

| Potential endogenous interferent | 25(OH)D concentration of non-spiked sample (ng/mL) | Highest Test Concentration that demonstrated no significant interference |
| --- | --- | --- |
| Rheumatoid Factor | 32.6 and 54.9 | 600 IU/mL |

Cholesterol, Total: Interference from total cholesterol was assessed using a recovery study. A serum sample containing 343.4 mg/dL cholesterol was spiked with 10% or 20% of a high serum pool containing 168.0 ng/mL 25(OH)D. Serum samples containing 392.6 mg/dL or 309.1 mg/dL cholesterol were spiked with 10% or 20% of a high serum pool containing 392.6 mg/mL 25(OH)D. Each sample was tested in replicates of 4. Recovery of 25(OH)D was assessed by comparing observed versus expected 25(OH)D values contributed by the 25(OH)D spike. Non-significant interference is defined as % recovery between 90-110%. The results are summarized in the following table:

| Potential endogenous interferent | 25(OH)D concentration of non-spiked sample(ng/mL) | Highest Test Concentration that demonstrated no significant interference |
| --- | --- | --- |
| Cholesterol, Total | 8.6 | 300 mg/dL |

{11}

The Sponsor has the following limitations in the labeling based on the interference studies:

- The lowest Bilirubin level that does not significant interfere (<10% bias) with the assay is 20 mg/dL; greater than +10% bias was first observed when bilirubin is above 22 mg/dL.
- Rheumatoid factor at 800 IU/mL will cause a bias in 25(OH)D results up to -11%.
- Cholesterol at 343 mg/dL will cause a bias in 25(OH)D results up to -19%.

## Cross-reactivity

Two studies were conducted according to CLSI EP7-A2 to evaluate the potential cross-reactivity of the assay with endogenous cross-reactants (25(OH)D₂ and 25(OH)D₃) and exogenous substances similar in chemical structure to 25(OH)D.

Endogenous cross-reactants 25(OH)D₂ and 25(OH)D₃ were spiked into serum samples, with initial concentrations of endogenous 25(OH)D₂, 25(OH)D₃, and 24, 25(OH)₂D₃ determined using an ID-LCMS/MS method. Spiked and un-spiked sample concentrations were measured with the IDS-iSYS 25VitDᵃ assay. The % cross-reactivity was calculated based on following equation:

$$
\frac{(\text{Mean conc. of spiked sample} - \text{mean conc. of unspiked sample}) \times 100}{\text{Spiked concentration}}
$$

## Cross reactivity results

| Potential Cross Reactant | Spike concentration (ng/mL) | % Cross Reactivity |
| --- | --- | --- |
| 25(OH)D₃ | 20 | 101 |
| 25(OH)D₂ | 20 | 105 |

Exogenous synthetic 25(OH)D metabolites were spiked into serum samples and spiked and un-spiked sample concentrations were measured with the IDS-iSYS 25VitDᵃ assay. The % cross reactivity was calculated based on the following equation:

$$
\frac{(\text{Mean conc. of spiked sample} - \text{mean conc. of unspiked sample}) \times 100}{\text{Spiked concentration}}
$$

## Cross reactivity results

| Potential Cross Reactant | Spike concentration (ng/mL) | % Cross Reactivity |
| --- | --- | --- |
| 24,25 dihydroxyvitamin D₃ | 25 | 197 |
| 24,25 dihydroxyvitamin D₂ | 25 | 37 |
| 1,25 dihydroxyvitamin D₃ | 20 | 3 |
| 1,25 dihydroxyvitamin D₂ | 20 | 8 |
| 3-epi-25-OH vitamin D₃ | 100 | 0 |

{12}

| Potential Cross Reactant | Spike concentration (ng/mL) | % Cross Reactivity |
| --- | --- | --- |
| 3-epi-25-OH vitamin D2 | 100 | 2 |
| Paricalcitol | 100 | -2 |
| Alfacalcidol | 500 | 0 |
| 25,26 dihydroxyvitamin D3 | 10 | 54 |
| Cholecalciferol (Vitamin D3) | 1000 | 1 |
| Ergocalciferol (Vitamin D2) | 100 | 5 |

f. Assay cut-off:

Not applicable.

2. Comparison studies:

a. Method comparison with predicate device:

A method comparison study was performed to compare the candidate device to the predicate device in accordance with CLSI EP9-A2. A total of 120 serum samples with a sample range of 7.3 to 112 ng/mL were tested in singlicate on the candidate and predicate devices. Of the 120 samples tested, 2 samples were altered (one sample was diluted and one sample was spiked) in order to cover the claimed measuring range. Passing-Bablok regression analysis was performed on the comparative data:

| n | Slope (ng/mL) [95% CI] | Intercept (ng/mL) [95% CI] | Correlation Coefficient (r) | Sample range tested (ng/mL) |
| --- | --- | --- | --- | --- |
| 120 | 0.93 [0.86 to 1.01] | 1.09 [-0.90 to 3.01] | 0.95 | 7.3 to 112 |

The candidate device (IDS-iSYS 25 VitD assay) is a modification of the predicate device (IDS-iSYS 25-Hydroxy Vitamin D assay (k140554)) and the main differences are the source of antibody pools and the calibrator matrix used. It is expected that the candidate device has a slightly different performance at the low end of the measuring range when compared against the predicate device because of the overall assay optimization. The slope showed an overall negative bias when compared against the predicate device. In addition, scattering above 60 ng/mL was also observed for this comparison study, but falls within the clinically insignificant range. The accuracy of the candidate device has been established through a method comparison study against the ID-LC-MS/MS 25(OH)D RMP and was the basis of the substantial equivalence determination.

An additional method comparison study was performed to compare the candidate device IDS-iSYS 25 VitD assay to the ID-LC-MS/MS 25(OH)D RMP. A total of 136 independent serum samples value assigned by ID-LC-MS/MS RMP (NIST/Ghent University) were tested. Of these 136 samples, 7 samples with a high 25(OH)D

{13}

concentration were diluted in a zero 25(OH)D matrix down to target values falling within the high end of the measuring range. The remaining 129 samples were unaltered. Passing Bablok regression analysis was performed on the comparative data:

| n | Slope (ng/mL) [95% CI] | Intercept (ng/mL) [95% CI] | Correlation Coefficient (r) | Sample range tested (ng/mL) |
| --- | --- | --- | --- | --- |
| 136 | 0.99 [0.94 to 1.05] | -0.51 [-1.93 to 0.75] | 0.97 | 5.6-110 |

# b. Matrix comparison:

A matrix comparison study using the IDS-iSYS $25\mathrm{VitD}^{\mathrm{S}}$ assay was performed according to CLSI EP09-A3 to evaluate the difference between serum (serum without additives, SST) and plasma (lithium heparin, sodium heparin, $\mathbf{K}_2$ EDTA). Sixty-seven (67) samples, ranging from 4.8 to $108~\mathrm{ng / mL}$ , were tested with ten of these samples spiked in order to cover the measuring range. Passing-Bablok regression analysis was performed on the comparative data relative to serum with no additives:

| Sample Type | Slope (ng/mL) [95% CI] | Intercept (ng/mL) [95% CI] | R2 |
| --- | --- | --- | --- |
| Serum – SST | 0.98 [0.94 to 1.02] | 0.18 [-0.31 to 1.12] | 0.99 |
| Plasma – K2EDTA | 0.96 [0.94 to 0.98] | 0.05 [-0.31 to 0.59] | 1.00 |
| Plasma – Lithium Heparin | 0.98 [0.93 to 1.01] | 0.22 [-0.52 to 0.86] | 1.00 |
| Plasma – Sodium Heparin | 0.99 [0.95 to 1.02] | 0.05 [-0.66 to 0.71] | 0.99 |

The results of the matrix comparison study support the sponsor's claim that serum as well as $\mathrm{K}_2$ EDTA, Lithium Heparin, and Sodium Heparin are acceptable sample types for this device.

# 3. Clinical studies:

a. Clinical Sensitivity:

Not applicable.

b. Clinical specificity:

Not applicable.

{14}

c. Other clinical supportive data (when a. and b. are not applicable):

Not applicable.

4. Clinical cut-off:

Not applicable.

5. Expected values/Reference range:

An expected value study was performed according to the non-parametric method in CLSI C28-A3c. Samples were collected from 392 apparently healthy male (51%) and female (49%) adults, aged 21-89 years, during winter and summer seasons and from geographically diverse regions of the United States. This overall diversity in, geographic location, race, and ethnicity represents a broad spectrum of UV light exposure in the intended use population. The samples were tested for 25(OH)D concentrations using the IDS-iSYS 25 VitD⁵ assay. The 2.5ᵗʰ to 97.5ᵗʰ reference interval is shown below:

25-OH vitamin D expected values for adults: 10.4 to 59.5 ng/mL (n = 392)

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.

15