25-Hydroxy Vitamin Ds EIA

K142351 · Immunodiagnostic Systems , Ltd. · MRG · Aug 25, 2015 · Clinical Chemistry

Device Facts

• Record ID: K142351
• Device Name: 25-Hydroxy Vitamin Ds EIA
• Applicant: Immunodiagnostic Systems , Ltd.
• Product Code: MRG · Clinical Chemistry
• Decision Date: Aug 25, 2015
• Decision: SESE
• Submission Type: Traditional
• Regulation: 21 CFR 862.1825
• Device Class: Class 2

Intended Use

The 25-Hydroxy Vitamin D⁵ EIA kit is intended for the quantitative determination of 25-hydroxyvitamin D [25(OH) D] and other hydroxylated metabolites in human serum or plasma. 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.

Device Story

The 25-Hydroxy Vitamin DS EIA is a manual enzyme immunoassay (EIA) for quantifying 25(OH)D and hydroxylated metabolites in human serum or plasma. The assay is performed in a microtitre plate by the user. Samples are diluted with biotin-labeled 25(OH)D and incubated in wells coated with sheep anti-25(OH)D antibody. Horseradish peroxidase (HRP)-labeled avidin is added, binding to the biotin-complexed 25(OH)D. After washing, a chromogenic substrate (TMB) is added. The resulting color intensity, measured via a microtitre plate reader, is inversely proportional to the 25(OH)D concentration. The device is used in clinical laboratory settings by trained personnel. Results assist clinicians in assessing vitamin D sufficiency, aiding in patient management and treatment decisions.

Clinical Evidence

Bench testing only. Precision evaluated per CLSI EP5-A2 (n≥80 per sample). Linearity (6.5-100 ng/mL) per CLSI EP6-A (R²=1.00). LoB/LoD/LoQ determined per CLSI EP17-A (LoQ 4.8 ng/mL). Interference and cross-reactivity studies performed. Method comparison against predicate (n=195) showed bias due to recalibration. Accuracy validated against Ghent/CDC ID-LC/MS/MS RMP (n=109, Deming regression y=0.97x-0.84, r=0.947). Reference range established using 280 healthy adults (12.3-49.0 ng/mL).

Technological Characteristics

Quantitative EIA; sheep polyclonal antibody-coated polystyrene microtiter plates; horseradish peroxidase-avidin conjugate; TMB/H2O2 substrate. Requires microplate colorimeter (450 nm). Traceable to ID-LC/MS/MS RMP and NIST SRM 2972. Compatible with serum, EDTA, lithium/sodium heparin, and sodium citrate plasma. Storage 2-8°C.

Indications for Use

Indicated for the quantitative determination of 25-hydroxyvitamin D and other hydroxylated metabolites 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

• OCTEIA 25-OH Vitamin D EIA (K021163)

Submission Summary (Full Text)

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Image /page/0/Picture/1 description: The image shows the logo for the U.S. Department of Health & Human Services. The logo consists of a stylized image of an eagle with its wings spread, and the text "DEPARTMENT OF HEALTH & HUMAN SERVICES - USA" arranged in a circular pattern around the eagle. The eagle is depicted in a simple, line-art style, and the text is in a sans-serif font.

August 25, 2015

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

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

Re: K142351

Trade/Device Name: 25-Hydroxy Vitamin De EIA Regulation Number: 21 CFR 862.1825 Regulation Name: Vitamin D test system Regulatory Class: II Product Code: MRG Dated: August 14, 2015 Received: August 19, 2015

Dear 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/Resourcesfor You/Industry/default.htm. Also, please note the regulation entitled. "Misbranding by reference to premarket notification" (21 CFR Part 807.97). For questions regarding the reporting of adverse events under the MDR regulation (21 CFR Part 803), please go to

http://www.fda.gov/MedicalDevices/Safety/ReportaProblem/default.htm for the CDRH's Office of Surveillance and Biometrics/Division of Postmarket Surveillance.

You may obtain other general information on your responsibilities under the Act from the Division of Industry and Consumer Education at its toll-free number [phone] or [phone] or at its Internet address

http://www.fda.gov/MedicalDevices/ResourcesforYou/Industry/default.htm.

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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# Indications for Use

510(k) Number (if known) k142351

Device Name 25-Hydroxy Vitamin DS EIA

:

#### Indications for Use (Describe)

The 25-Hydroxy Vitamin DS EIA assay is intended for the quantitative determination of 25-hydroxyvitamin D [25(OH)D] and other hydroxylated metabolites in human serum or plasma. 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.

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

X Prescription Use (Part 21 CFR 801 Subpart D)

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

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| 510k Number | 510(k) SUMMARY<br>k142351 | |
|----------------------------------------------------------------------|------------------------------------------------------------------------------------------------------------------------------------|-------------------------------------------------------------------------------------------------------------------------------------|
| Introduction | determination of substantial equivalence. | According to the requirements of 21CFR807.92, the following<br>information provides sufficient detail to understand the basis for a |
| Submitter | Immunodiagnostic Systems Limited<br>10 Didcot Way<br>Boldon Business Park<br>Boldon<br>Tyne and Wear<br>NE35 9PD<br>United Kingdom | |
| | Contact Person: Mick Henderson<br>Phone: [phone]<br>Fax: [phone]<br>Email: [e-mail] | |
| | Secondary Contact: Mr. R. Prebula<br>Hogan Lovells US LLP,<br>Columbia Square,<br>555 Thirteenth Street, NW,<br>Washington DC | |
| | 20004<br>Washington<br>Phone: [phone]<br>Fax: [phone]<br>Email: [e-mail] | |
| | Date prepared:18 August 2015 | |
| Device Name | | |
| | Proprietary names: | 25-Hydroxy Vitamin Dª EIA |
| | Common names: | Vitamin D test |
| | Classification:<br>Regulatory Class: | 21CFR862.1825 Vitamin D Test System<br>II |
| | Product Code: | MRG |
| IDS, OCTEIA 25-OH Vitamin D EIA, (k021163)<br>Predicate Device Name: | | |

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# A. Test Principle:

The 25-Hydroxy Vitamin De EIA is a manual assay test and does not require the use of an automated system. The whole assay is performed in a microtitre plate and requires a user to perform each step.

# The 25-Hydroxy Vitamin De EIA assay

The 25-Hydroxy Vitamin De assay is an enzymeimmunoassay for the quantitation of 25(OH)D and other hydroxylated metabolites in serum or plasma. 25uL of each calibrators, controls and samples are diluted with biotin labelled 25(OH)D. The diluted samples are incubated in microtitre wells which are coated with a highly specific sheep 25(OH)D at room temperature before aspiration and washing. Enzyme (horseradish peroxidase) labelled avidin, is added and binds selectively to complexed biotin and, following a further wash step, colour is developed using a chromogenic substrate (TMB).

The absorbance of the stopped reaction mixtures are read in a microtitre plate reader, colour intensity developed being inversely proportional to the concentration of 25(OH)D.

# B. Indications For Use:

For In Vitro Diagnostic Use

The 25-Hydroxy Vitamin De EIA assay is intended for the quantitative determination of 25-hydroxyvitamin D [25(OH)D] and other hydroxylated metabolites in human serum or plasma. 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.

Rx Only

# C. Comparison with the predicate:

Similarities compared to the chosen predicate device (OCTEIA 25-hydroxy vitamin D EIA)

| | Predicate<br>K021163 | Candidate Device |
|---------------------|---------------------------------------------------------------------------------------------------------------------------------------------------------|------------------|
| Intended Use | The OCTEIA 25-hydroxy<br>vitamin D EIA (25-OH D) kit is<br>an assay for the quantitative<br>measurement of 25-Hydroxy<br>vitamin D in serum and plasma. | Same |
| Indications for use | Results are to be used in<br>conjunction with other clinical<br>and laboratory data to assist the | Same |

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| | clinician in the assessment of<br>vitamin D sufficiency | |
|----------------------|----------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------|------|
| Analyte | 25-hydroxy vitamin D | Same |
| Reagent storage | 2-8°C | Same |
| Capture | Antibody coated micro plate | Same |
| Calibrator | Low charcoal stripped human<br>serum containing 25-hydroxy<br>vitamin D and sodium azide as a<br>preservative | Same |
| Sample volume | 25µL | Same |
| Units | nmol/L | Same |
| Kit components | EIA kit consisting of Anti-25-<br>OH D coated plate, Biotinylated<br>25-OH D, Standards (7 levels),<br>kit controls (2 levels), Avidin<br>HRP solution, substrate solution,<br>stop solution and wash solution | Same |
| Calibration | Full standard curve to be run<br>with all assays | Same |
| Calibration interval | Per assay run | Same |
| Quality Control | Two (2) controls provided | Same |
| Conversion of units | nmol/L x 0.40 = ng/mL<br>ng/ml. x 2.5 = nmol/L | Same |

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

| | Predicate<br>K021163 | Candidate Device |
|-----------------------------------------------------------|------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------|-----------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------|
| Antibodies | Sheep anti 25-hydroxy vitamin<br>D. | new source |
| Analytical sensitivity | 5 nmol/L | NA |
| Functional Sensitivity<br>(Limit of Quantitation<br>(LoQ) | NA | 4.8ng/mL (12 nmol/L) |
| Limit of Detection<br>(LoD) | NA | 2.7ng/mL (6.9 nmol/L) |
| Limit of Blank<br>(LoB) | NA | 1.3ng/mL (4.5nmol/L) |
| Precision | Intra-assay Precision n = 10<br>5.3% to 6.7% in the<br>concentration range 39 to<br>165nmol/L<br>Inter-assay Precision n = 11<br>4.6% to 8.7% in the<br>concentration range 40 to<br>132nmol/L | Within Run Precision n = 88<br>1.9% to 3.7% in the<br>concentration range 11.7 to<br>65.1 ng/mL (29.3 to 183<br>nmol/L)<br>Total Precision n = 88<br>3.7% to 11.6% in the<br>concentration range 11.7 to<br>65.1ng/mL (29.3 to 183<br>nmol/L) |

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| Specificity, | Specificity | Specificity |
|------------------------|-----------------------|-------------------------------------------------------------------------------------------------------------------------------------------------------------------------------|
| Interfering substances | 25-Hydroxy vitamin D3 | 25-Hydroxy vitamin D3 |
| & Cross Reactivity | 100% | તે તે જેની જેવી સવલતો પ્રાપ્ય થયેલી છે. આ ગામનાં લોકોનો મુખ્ય વ્યવસાય ખેતી, ખેતમજૂરી તેમ જ પશુપાલન છે. આ ગામનાં મુખ્યત્વે ખેતી, ખેતમજૂરી તેમ જ પશુપાલન છે. આ ગામનાં મુખ્યત્વે |
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| | | |

| Performance | Predicate<br>K021163 | Candidate Device |
|--------------------------------------------------------------|-------------------------------------------------------------------------------------------------------------------------------------------------------------|-----------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------|
| Specificity,<br>Interfering substances<br>& Cross Reactivity | Interference:<br>Haemoglobin<br>Tested up to 1470 mg/dL<br><br>Bilirubin<br>Tested up to 513 µmol/L<br><br>Lipid<br>Tested up to 5.6 mmol/L<br>triglyceride | Interference:<br>Haemoglobin<br>No interference up to<br>400mg/dL<br><br>Bilirubin, conjugated<br>No interference up to 20mg/dL<br><br>Triglyceride (Intra Lipid)<br>No interference up to<br>475mg/dL<br><br>Total Protein<br>No interference up to 9.2g/dL<br><br>HAMA<br>No interference up to<br>1000ng/mL<br><br>Red Blood Cells<br>No interference up to 0.4%<br><br>Rheumatoid Factor<br>No interference up to<br>800IU/mL<br><br>Vitamin D Binding Protein<br>(Gc globulin)<br>No interference up to 2000<br>ng/mL<br><br>Cholesterol, Total<br>No interference up to<br>500mg/dL<br><br>Biotin<br>No interference up to<br>200nmol/L |
| Performance | Predicate<br>K021163 | Candidate Device |
| | Cross Reactivity<br>25-Hydroxy vitamin D2<br>75% | Cross Reactivity<br>25-Hydroxy vitamin D2<br>109% |
| | 24,25-DiHydroxy vitamin D3<br>$\ge$ 100% | 24-25 Di- Hydroxy vitamin D3<br>95% |
| | | 24,25-(OH)2D3<br>91% |
| | Cholecalciferol (D3)<br>< 0.01% | 3-epi-25-OH Vitamin D3<br>-1% |
| | Cholecalciferol (D2)<br>< 0.30% | 3-epi-25-OH Vitamin D2<br>-1% |
| | | 1,25-(OH)2D3<br>5% |
| | | 1,25-(OH)2D2<br>84% |
| | | Paricalcitol<br>17% |
| | | 25(OH)D3<br>95% |
| | | 25(OH)D2<br>109% |
| | | Vitamin D3 (Cholecalciferol)<br>0% |
| | | Vitamin D2 (Ergocalciferol)<br>6% |
| Performance | Predicate<br>K021163 | Candidate Device |
| Reference range/<br>Expected Values | 47.7 – 144 nmol/L | Non Supplemented<br>11.2 to 45.9ng/mL<br>28.0 to 114.6 nmol/L<br>Supplemented<br>15.4 to 86.8ng/mL<br>38.5 to 217.1 nmol/L<br>Overall<br>12.3 to 49ng/mL<br>30.7 to 122.5 nmol/L |
| Method comparison | Against a recognised<br>radioimmunoassay<br>n = 180<br>AC57 = 1.01(x) + 0.7<br>Correlation coefficient (r) =<br>0.9 | Against Predicate device<br>n = 195<br>Passing Bablok regression:<br>25-Hydroxy Vitamin DS= 0.88<br>x (25-Hydroxy Vitamin D) +<br>3.2 ng/mL (+ 8.1 nmol/L)<br>Pearson correlation coefficient,<br>r: 0.94 |
| Reportable Range | 2ng/mL to 152ng/mL | 6.5 to 100ng/mL |
| Linearity | Mean Measured / Expected<br>102%<br>Range individual dilutions<br>88% to 125% | Linear regression of the<br>observed concentrations versus<br>the expected concentrations:<br>Observed = 1.02 x (Expected)<br>+ 0.23 ng/mL<br>Observed = 1.02 x (Expected)<br>+ 0.58 nmol/L<br>Regression coefficient R²:=<br>1.00<br>Maximum deviation; -8.8% for<br>samples > 20 ng/mL and 2.37<br>ng/ml for samples < 20 ng/mL |
| Sample matrix<br>(primary tube type) | Serum or Plasma (EDTA or<br>Heparin) | Serum (standard sampling<br>tubes or tubes containing<br>serum separating gel) or<br>plasma (EDTA, lithium<br>heparin, sodium heparin or<br>sodium citrate) |

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#### D. Performance Characteristics :

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

Precision was determined in accordance with CLSI EP5-A2, "Evaluation of Precision Performance of Quantitative Measurement Methods". 10 serum samples were assayed using 3 lots of reagents in duplicate, twice per day for a minimum of 20 days (n ≥ 80 replicates per sample).

| ng/mL | | | | | | | | | | |
|-------|--------------|------|------------|------|-------------|-------|-------------|------|-------|-------|
| | EP Evaluator | | | | | | | | | |
| | | | Within Run | | Between Run | | Between Day | | Total | |
| | n = | Mean | SD | %cv | SD | %cv | SD | %cv | SD | %cv |
| IQC1 | 88 | 19.1 | 0.4 | 1.9% | 0.6 | 3.2% | 0.1 | 0.4% | 0.7 | 3.7% |
| IQC2 | 88 | 43.7 | 0.8 | 1.9% | 0.9 | 4.3% | 0.0 | 0.0% | 2.0 | 4.6% |
| IQC3 | 88 | 65.1 | 2.3 | 3.5% | 4.0 | 6.2% | 1.7 | 2.6% | 4.9 | 7.6% |
| S1 | 88 | 11.7 | 0.4 | 3.4% | 1.3 | 11.0% | 0.0 | 0.0% | 1.4 | 11.5% |
| S2 | 88 | 24.5 | 0.6 | 2.5% | 1.3 | 5.3% | 0.0 | 0.0% | 1.4 | 5.8% |
| S3 | 88 | 40.7 | 1.0 | 2.5% | 2.1 | 5.1% | 0.0 | 0.0% | 2.3 | 5.7% |
| S4 | 88 | 73.2 | 2.7 | 3.7% | 4.0 | 5.5% | 0.6 | 0.8% | 4.9 | 6.6% |
| S5 | 88 | 21.7 | 0.5 | 2.4% | 0.9 | 4.1% | 0.0 | 0.0% | 1.0 | 4.7% |
| S6 | 88 | 51.0 | 1.9 | 3.7% | 3.4 | 6.6% | 0.0 | 0.0% | 3.9 | 7.6% |
| S7 | 88 | 28.3 | 0.8 | 2.7% | 1.7 | 6.2% | 0.0 | 0.0% | 1.9 | 6.7% |

Results from one representative lot is summarized in the table below (n=88)

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

Linearity was evaluated based on a modified version of CLSI EP-6A, "Evaluation of the Linearity of Quantitative Measurement Procedures". Samples containing varying concentrations of 25-hydroxyvitamin D were assayed in replicate of four. The resulting mean concentrations were compared to predicted concentrations. Samples were prepared by diluting a high patient sample with a low patient sample prior to assay. The linear regression (weighted) of the Observed concentrations versus the expected concentrations is:

Observed = 1.02 x (Expected) + 0.23 ng/mL Observed = 1.02 x (Expected) + 0.58 nmol/L Regression coefficient R == 1.00 Maximum deviation; -8.8% for samples > 20 ng/mL and 2.37 ng/ml for samples < 20 ng/mL

The reportable range of the assay is 6.5 - 100 ng/mL (16.3 - 250 nmol/L). Any value that reads below 6.5 ng/mL (16.3nmol/L) should be reported as "< 6.5 ng/mL (16.3 nmol/L)".

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

The 25-Hydroxy Vitamin D assay is traceable to the isotope dilution-liquid chromatography/tandem mass spectrometry (ID-LCMS/MS) 25(OH) Vitamin D reference method procedure (RMP) which was used in assigning the target value for the Vitamin D Standardization Program (VDSP single donor human serum panel. The ID-LCMS/MS RMP is traceable to the National Institute of Standards and Technology Standard Reference Material (SRM) 2972.

The 25-Hydroxy Vitamin DS assay is certified by the CDC Vitamin D Standardization Certification Program (VDSP) http://www.cdc.gov/labstandards/hs.html

## Stability

This kit is stable for 8 months when stored at 2-8℃.

| Kit component | After opening or preparation (open vial stability) |
|-------------------------------|-------------------------------------------------------------------------------------|
| Biotin 25(OH)D Solution | 8 weeks<br>after reconstitution<br>Store at 2-8°C in the dark immediately after use |
| Unused Ab. coated plate strip | 8 weeks<br>Store at 2-8°C in foil pouch with<br>desiccant sachet |
| Calibrators,<br>Controls | 8 weeks<br>Store at 2-8°C after opening |
| Wash Solution | 8 weeks<br>Store at room temperature (18-25 °C) after preparation |

#### d. Detection limit:

The limit of blank (LoB), limit of detection (LoD) and limit of quantitation (LoQ) were determined based on guidance from CLSI EP17-A "Protocols for the determination of limits of detection and limits of quantitation".

| Sensitivity | 25OHD Levels |
|-------------|-------------------------|
| LoB | 1.3 ng/mL (4.5 nmol/L) |
| LoD | 2.7 ng/mL (6.9 nmol/L) |
| LoQ | 4.8 ng/mL (12.0 nmol/L) |

e. Analytical specificity:

Cross-reactivity of endogenous 25(OH) vitamin D metabolite. Endogenous 25(OH) vitamin D metabolites were spiked into vitamin D serum samples and analyzed with the 25-Hydroxy Vitamin Do assay.

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| Cross Reactant | Spike Conc. (ng/mL) | Mean % Cross Reactivity |
|----------------|---------------------|-------------------------|
| 25(OH)D3 | 10.0 | 95% |
| | 20.0 | |
| 25(OH)D2 | 10.0 | 109% |
| | 20.0 | |
| 24,25-(OH)2D3 | 4.0 | 91% |
| | 6.0 | |

Cross-reactivity of exogenous synthetic 25(OH) vitamin D metabolite. Exogenous synthetic 25(OH) vitamin D metabolites were spiked into vitamin D serum samples and analyzed with the 25-Hydroxy Vitamin De assay.

| Cross Reactant | Spike Conc.<br>(ng/mL) | Mean% Cross<br>Reactivity |
|-----------------------------|------------------------|---------------------------|
| 3-epi-25(OH) D3 | 100 | -1% |
| 3-epi-25(OH) D2 | 100 | -1% |
| 1,25-(OH)2D3 | 2 | 5% |
| 1,25-(OH)2D2 | 20 | 84% |
| VitaminD3 (Cholecalciferol) | 1000 | 0% |
| VitaminD2 (Ergocalciferol) | 100 | 6% |
| Paricalcitol* | 100 | 17% |

* Paricalcitol interferes with the 25-Hydroxy Vitamin De EIA test. Paricalcitol, when tested, caused a positive bias in sample result.

The following substances do not interfere in the 25-Hydroxy Vitamin DS EIA assay when the concentrations presented in the following table are below the stated threshold.

| Potentially Interfering | Threshold |
|-------------------------------|---------------|
| Agent | Concentration |
| Triglycerides | 475 mg/dL |
| Bilirubin (conjugated) | 20 mg/dL |
| Haemoglobin | 400 mg/dL |
| HAMA | 1000 ng/mL |
| Rheumatoid Factor | 800 IU/mL |
| Red Blood Cells | 0.40% |
| Vitamin D Binding<br>Proteins | 2000 ng/dL |

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| Total Protein | 9.2g/dL |
|--------------------|-----------|
| Cholesterol, Total | 500 mg/mL |
| Biotin | 200nmol/L |

- 2. Comparison studies:
- a. Method comparison with predicate device:

The 25-Hydroxy Vitamin DS EIA Assay was compared against the 25-Hydroxy Vitamin D EIA assay (K021163) for the quantitative determination of 25-Hydroxy Vitamin D (and other hydroxylated metabolites), following CLSI EP-9A2, "Method Comparison and Bias Estimation Using Patient Samples".

Correlation to 25-Hydroxy Vitamin D EIA, AC57 (k021163)

| Passing Bablok | $y = 0.88x + 3.23$ ng/mL | $y = 0.88x + 8.05$ nmol/L |
|------------------------------------|--------------------------|----------------------------|
| Slope, 95% Confidence Interval | 0.86 to 0.91 | 0.86 to 0.91 |
| Intercept, 95% Confidence Interval | 2.71 to 3.83 ng/mL | 6.85 to 9.45 nmol/L |
| Linear Regression | $y = 0.82x + 4.86$ ng/mL | $y = 0.82x + 12.35$ nmol/L |
| Slope, 95% Confidence Interval | 0.80 to 0.84 | 0.80 to 0.84 |
| Intercept, 95% Confidence Interval | 4.14 to 5.57 ng/mL | 10.35 to 13.92 nmol/L |
| Correlation Coefficient, r | 0.97 | 0.97 |
| n | 195 | 195 |
| Difference Plot | -0.61 | -1.53 |

Additional method comparison study was performed against the reference measurement procedure using 109 in dependent native serum samples which has been value assigned by the Ghent reference method procedure. Regression analysis was summarized below:

| | ng/mL | nmol/L |
|------------------------------------|--------------------------|---------------------------|
| Passing Bablok | $y = 0.96x - 0.11$ ng/mL | $y = 0.96x - 0.34$ nmol/L |
| Slope, 95% Confidence Interval | 0.88 to 1.02 | 0.88 to 1.02 |
| Intercept, 95% Confidence Interval | -1.62 to 1.88 ng/mL | -4.15 to 4.68 nmol/L |
| Deming Regression | $y = 0.97x - 0.84$ ng/mL | $y = 0.97x - 2.07$ nmol/L |
| Slope, 95% Confidence Interval | 0.90 to 1.04 | 0.90 to 1.04 |
| Intercept, 95% Confidence Interval | -2.75 to 1.08 ng/mL | -6.85 to 2.71 nmol/L |
| Correlation Coefficient, r | 0.947 | 0.947 |
| n | 109 | 109 |
| Diffrence Plot | -1.77 | -4.42 |

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## b. Matrix comparison:

Summary of the statistical methods used for alternative blood tube types to serum blood tube using a paired tube comparison is shown below.

Passing-Bablok analysis of the test and comparator assays was performed, taking the slope, intercept and correlation coefficient (with 95 % confidence intervals) and difference bias plot.

| | SST | EDTA | Sodium Heparin |
|------------------------------------|---------------------|---------------------|---------------------|
| n | 28 | 38 | 28 |
| Passing bablok | $y=0.99x+0.18 g/mL$ | $y=0.97x+0.60ng/mL$ | $y=1.07x-0.47ng/mL$ |
| Slope,95% confidence level | 0.95 to 1.03 | 1.00 to 1.01 | 1.00 to 1.10 |
| Intercept, 95% confidence<br>level | -0.17 to 0.65ng/mL | -0.15 to 1.04ng/mL | -1.21 to 0.68ng/mL |
| Correlation Coefficient r | 0.99 | 1.00 | 0.99 |
| Mean Bias | 0.41 | 0.07 | 1.11 |

| | Lithium Heparin | Citrate |
|---------------------------------|---------------------|---------------------|
| n | 28 | 28 |
| Passing Bablok | Y=1.04x -0.22 ng/mL | Y=1.03x -0.70 ng/mL |
| Slope, 95% confidence level | 1.01 to 1.08 | 0.98 to 1.06 |
| Intercept, 95% Confidence level | -0.73 to 0.60 ng/mL | -1.36 to 0.49 ng/mL |
| Correlation Coefficient, r | 1.00 | 0.99 |
| Mean Bias | 1.50 | 0.43 |

The design criterion was that the slope must be 0.85 to 1.15 and the intercept +/- 7 ng/mL and r ≥0.90.

Serum separator tubes (SST), EDTA plasma, Sodium Heparin plasma, Lithium Heparin plasma and citrate plasma tube data do not present any new issues of safety or effectiveness for the 25-Hydroxy Vitamin De EIA assay.

# 3. Expected values/Reference range:

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

Samples from 280 apparently light skin and dark skin healthy male adults (71.1%) and female adults (28.9%) aged 21-77 years living 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 25-Hydroxy Vitamin Do Assay. Samples were from individuals with normal values for intact PTH, calcium, phosphate, and TSH, and not taking any interfering medications. The following ranges were determined using the 25-Hydroxy Vitamin De assay and are provided for information only. The 95 % reference interval for apparently healthy adults, were calculated by a non-parametric

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

method following guidance from CLSI C28-A3 " Defining, Establishing and Verifying Reference Intervals in the Clinical Laboratory".

Obtained normal adults range:12.3 to 49.0 ng/mL (30.7 to 122.5 nmol/L) (n=280)

| | n | % | Observed Sample Range<br>(ng/mL) | Median<br>(ng/mL) |
|------------------|-----|-------|----------------------------------|-------------------|
| Non-supplemented | 219 | 78.2% | 11.2 to 45.9 | 25.1 |
| Northern US | 116 | 41.4% | 10.3 to 35.1 | 22.6 |
| Southern US | 103 | 36.8% | 14.6 to 48.0 | 29.5 |
| Supplemented | 61 | 21.8% | 15.4 to 86.8 | 30.2 |
| Overall | 280 | 100% | 12.3 to 49.0 | 26.0 |

Observed sample ranges were:

| | n | % | Observed Sample Range<br>(nmol/L) | Median<br>(nmol/L) |
|------------------|-----|-------|-----------------------------------|--------------------|
| Non-supplemented | 219 | 78.2% | 28.0 to 114.6 | 62.8 |
| Northern US | 116 | 41.4% | 25.6 to 87.6 | 56.5 |
| Southern US | 103 | 36.8% | 36.6 to 120.0 | 73.9 |
| Supplemented | 61 | 21.8% | 38.5 to 217.1 | 75.5 |
| Overall | 280 | 100% | 30.7 to 122.5 | 65.0 |

The above ranges should be considered as guidelines only; it is recommended that each laboratory establish its own expected range based for its own patient population.

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

12.3 to 49.0 ng/mL (n = 280) Normal Adults 30.7 to 122.5 nmol/L (n = 280)

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 250HD.

## E. Conclusion:

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