ST AIA-PACK 25-OH Vitamin D, ST AIA-PACK 25-OH Vitamin D Calibrator Set, AIA-PACK 25-OH Vitamin D Control Set, and ST AIA-PACK 25-OH Vitamin D Pretreatment Set

K150270 · Tosoh Bioscience, Inc. · MRG · Oct 26, 2015 · Clinical Chemistry

Device Facts

• Record ID: K150270
• Device Name: ST AIA-PACK 25-OH Vitamin D, ST AIA-PACK 25-OH Vitamin D Calibrator Set, AIA-PACK 25-OH Vitamin D Control Set, and ST AIA-PACK 25-OH Vitamin D Pretreatment Set
• Applicant: Tosoh Bioscience, Inc.
• Product Code: MRG · Clinical Chemistry
• Decision Date: Oct 26, 2015
• Decision: SESE
• Submission Type: Traditional
• Regulation: 21 CFR 862.1825
• Device Class: Class 2

Indications for Use

ST AIA-PACK 25-OH Vitamin D is designed for in vitro diagnostic use only for the quantitative measurement of total 25-hydroxyvitamin D (25-OH vitamin D) in human serum, Na-heparinized plasma or EDTA plasma on TOSOH AIA System Analyzers. The Tosoh ST AIA-PACK 25-OH Vitamin D is intended as an aid in the determination of Vitamin D sufficiency. The ST AIA-PACK 25-OH Vitamin D Calibrator Set is intended for in vitro diagnostic use only for the calibration of the ST AIA-PACK 25-OH Vitamin D assay.

Device Story

Quantitative fluorescent immunoassay for total 25-hydroxyvitamin D; used on Tosoh AIA System Analyzers (e.g., AIA-2000). Input: human serum, Na heparinized or EDTA plasma. Process: sample pretreatment with sodium hydroxide dissociates 25-OH vitamin D from binding proteins; competitive binding reaction occurs on magnetic beads coated with anti-25-OH vitamin D sheep monoclonal antibody; enzyme-labeled 25-OH vitamin D competes for binding sites; unbound components washed away; fluorogenic substrate (4MUP) added. Output: fluorescence signal inversely proportional to 25-OH vitamin D concentration; concentration calculated via standard curve. Used in clinical laboratories by trained personnel. Benefits: standardized measurement aligned with Vitamin D Standardization Program (VDSP) for improved accuracy in assessing Vitamin D status.

Clinical Evidence

Bench testing only. Precision evaluated per CLSI EP5-A2 (N=160, total CV 2.2-4.9%). Linearity confirmed per CLSI EP6-A (4-120 ng/mL, R^2=1.00). Detection limits (LoB 1.6, LoD 3.2, LoQ 3.3 ng/mL) established per CLSI EP17-A. Method comparison against Ghent ID-LC-MS/MS RMP (N=111) showed Weighted Deming slope 0.98, r=0.965. Reference range study (N=252) established 12.3-60.0 ng/mL for normal adults.

Technological Characteristics

Quantitative fluorescent immunoassay. Components: magnetic beads coated with sheep monoclonal anti-25-OH vitamin D antibody, enzyme-labeled 25-OH vitamin D conjugate, 4-methylumbelliferyl phosphate (4MUP) substrate. Instrumentation: Tosoh AIA System Analyzers. Traceability: ID-LC/MS/MS 25-OH vitamin D reference measurement procedure (Ghent University) traceable to NIST SRM 2972. Software: embedded instrument control and data processing.

Indications for Use

Indicated for quantitative measurement of total 25-hydroxyvitamin D in human serum, Na-heparinized plasma, or EDTA plasma to aid in determining 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

• ST AIA-PACK 25-OH Vitamin D Assay (k123131)

Submission Summary (Full Text)

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

A. 510(k) Number:
k150270

B. Purpose for Submission:
Modification of traceability for standardization of the previously cleared device, k123131

C. Measurand:
Total 25-hydroxyvitamin D (25-OH vitamin D)

D. Type of Test:
Quantitative fluorescent immunoassay

E. Applicant:
Tosoh Bioscience, Inc.

F. Proprietary and Established Names:
ST AIA-PACK 25-OH Vitamin D
ST AIA-PACK 25-OH Vitamin Calibrator Set

G. Regulatory Information:
1. Regulation section:
21 CFR 862.1825, Vitamin D Test System
21 CFR 862.1150, Calibrator
2. Classification:
Class II
3. Product code:
MRG-Vitamin D Test System
JIT-Calibrator, secondary

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4. Panel:

Clinical Chemistry (75)

H. Intended Use:

1. Intended use(s):

See indications for use below.

2. Indication(s) for use:

ST AIA-PACK 25-OH Vitamin D is designed for in vitro diagnostic use only for the quantitative measurement of total 25-hydroxyvitamin D (25-OH vitamin D) in human serum, Na heparinized or EDTA plasma on Tosoh AIA System Analyzers. The TOSOH ST AIA-PACK 25-OH Vitamin D is intended as an aid in the determination of Vitamin D sufficiency.

The ST AIA-PACK 25-OH Vitamin D Calibrator Set is intended for In Vitro Diagnostic Use Only for the calibration of the ST AIA-PACK 25-OH Vitamin D assay.

3. Special conditions for use statement(s):

For prescription use only

4. Special instrument requirements:

For use on the TOSOH AIA-2000 System Analyzer.

I. Device Description:

1. Tosoh's ST AIA-PACK 25-OH Vitamin D kit is provided as:

- 5 trays of 20 plastic test cups containing twelve magnetic beads coated with lyophilized anti-25-OH vitamin D sheep monoclonal antibody with sodium azide as a preservative.
- 5 vials containing 5 mL 25-OH vitamin D conjugated to bovine alkaline phosphatase.

2. Tosoh's ST AIA-PACK 25-OH Vitamin D Calibrator set is sold separately and contains: Six assigned concentrations of 25-OH Vitamin D in human serum (described on each vial) with sodium azide as preservative in aliquots of 1.0 mL, comes in lyophilized form, with vitamin D at target values of 0 (base material), 10, 20, 40, 80, and 165 ng/mL. The calibrator materials have been tested by FDA-approved methods and found negative for the presence of HBsAg, antibody to HIV-1/2 and HCV.

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J. Substantial Equivalence Information:

1. Predicate device name(s):
ST AIA-PACK 25-OH Vitamin D

2. Predicate 510(k) number(s):
k123131

3. Comparison with predicate:

| Similarities and differences for assay | | |
| --- | --- | --- |
| Item | Candidate device
ST AIA-PACK 25-OH Vitamin D
(k150270) | Predicate device
ST AIA-PACK 25-OH Vitamin D
(k123131) |
| Intended Use | ST AIA-PACK 25-OH Vitamin D is designed for in vitro diagnostic use only for the quantitative measurement of total 25-hydroxyvitamin D (25-OH vitamin D) in human serum, Na heparinized or EDTA plasma on TOSOH AIA System Analyzers. The Tosoh ST AIA-PACK 25-OH Vitamin D is intended as an aid in the determination of Vitamin D sufficiency. | Same |
| Sample Type | EDTA plasma, Na heparinized plasma, or serum | Same |
| Assay Methodology | Fluorescent immunoassay | Same |
| Assay Range | 4.0 – 120.0 ng/mL | Same |
| Incubation Time | 10 min cycle | Same |
| Traceability/Standardization | Traceable to the ID-LC/MS/MS 25 (OH) vitamin D reference measurement procedure (University of Ghent) via patient sample correlation. | Traceable to an Internal reference standard |
| Reference range | 12.3-60.0 ng/mL | 10.8 to 54.75 ng/mL |

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| Similarities and Differences for Calibrators | | |
| --- | --- | --- |
| Item | Candidate device
ST AIA-PACK 25-OH
Vitamin D Calibrator Set
(k150270) | Predicate device
ST AIA-PACK 25-OH
Vitamin D Calibrator Set
(k123131) |
| Intended Use | The ST AIA-PACK 25-OH Vitamin D Calibrator Set is intended for In Vitro Diagnostic Use Only for the calibration of the ST AIA-PACK 25-OH Vitamin D assay | Same |
| Matrix | Human Serum | Same |
| Format | Lyophilized | Same |
| Levels | Approximately 0-135 ng/mL (0, 8. 17, 33, 66, 135 ng/mL) | Approximately 0-165 ng/mL (0, 10, 20, 40, 80, 165 ng/mL) |
| Traceability/
Standardization | Traceable to the ID-LC/MS/MS 25 (OH) vitamin D reference measurement procedure (University of Ghent) via patient sample correlation. | Traceable to an Internal reference standard |

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

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

CLSI EP6-A: Evaluation of the Linearity of Quantitative Measurement Procedures: A Statistical Approach; Approved Guideline

CLSI C28-A3c: Establishing, and Verifying Reference Intervals in the Clinical Laboratory; Approved Guideline – Third Edition

L. Test Principle:

The ST AIA-PACK 25-OH Vitamin D is a one-step delayed competitive enzyme immunoassay which, after sample pretreatment, is performed entirely in the ST AIA-PACK 25-OH Vitamin D test cup. Sample pretreatment reagents (containing sodium hydroxide) disassociate 25-OH vitamin D from its binding proteins in the test sample. 25-OH vitamin D present in the pretreated sample is bound to 25-OH vitamin D-specific monoclonal antibody immobilized on magnetic beads. After that, the enzyme-labeled 25-OH vitamin D is added to the reaction mixture. The enzyme-labeled 25-OH vitamin D competes with 25-OH vitamin D for binding to the antibody on magnetic beads in the reaction mixture.

After the second incubation, the magnetic beads are washed to remove the unbound enzyme-

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labeled 25-OH vitamin D and are then incubated with a fluorogenic substrate, 4-methylumbelliferyl phosphate (4MUP). The amount of enzyme-labeled 25-OH vitamin D that binds to the beads is inversely proportional to the 25-OH vitamin D concentration in the test sample. A standard curve is constructed, and unknown 25-OH vitamin D concentrations are calculated using this curve.

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

### 1. Analytical performance:

#### a. Precision/Reproducibility:

A study was performed in accordance with CLSI EP5 A2 in which six (6) serum samples were assayed in duplicate in two runs per day for 20 non-consecutive days on two analyzers using two reagent lots for a total of 160 results per sample. Each analyzer/reagent lot produced similar precision results. The within-run and total precision results are summarized in the table below using one representative lot of reagents:

Representative lot precision:

| | | Within-run | | | Total | |
| --- | --- | --- | --- | --- | --- | --- |
| Serum sample | N | Mean (ng/mL) | SD | CV% | SD | CV% |
| 1 | 80 | 17.2 | 0.60 | 3.5 | 0.75 | 4.4 |
| 2 | 80 | 20.8 | 0.84 | 4.0 | 0.91 | 4.4 |
| 3 | 80 | 25.2 | 0.85 | 3.4 | 0.97 | 3.8 |
| 4 | 80 | 55.8 | 1.38 | 2.5 | 1.66 | 3.0 |
| 5 | 80 | 75.4 | 1.79 | 2.4 | 2.11 | 2.8 |
| 6 | 80 | 96.0 | 1.24 | 1.3 | 2.13 | 2.2 |

The combined lot precision is summarized in the table below:

| N=160 | Within run | | Between run | | Between day | | Between lot | | Total | |
| --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- |
| Mean (ng/mL) | SD | CV% | SD | CV% | SD | CV% | SD | CV% | SD | CV% |
| 16.9 | 0.62 | 3.7 | 0.53 | 3.1 | 0.64 | 3.8 | 0.53 | 3.1 | 0.82 | 4.9 |
| 20.9 | 0.82 | 3.9 | 0.65 | 3.1 | 0.68 | 3.3 | 0.36 | 1.7 | 0.96 | 4.6 |
| 24.7 | 0.77 | 3.1 | 0.52 | 2.1 | 1.02 | 4.1 | 0.76 | 3.1 | 1.19 | 4.8 |
| 55.2 | 1.28 | 2.3 | 1.07 | 1.9 | 1.48 | 2.7 | 1.16 | 2.1 | 1.81 | 3.3 |
| 74.6 | 1.55 | 2.1 | 1.26 | 1.7 | 1.88 | 2.5 | 1.57 | 2.1 | 2.28 | 3.1 |
| 95.3 | 1.24 | 1.3 | 1.40 | 1.5 | 2.07 | 2.2 | 1.03 | 1.1 | 2.43 | 2.6 |

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

Linearity was evaluated based on CLSI EP-6A, Evaluation of the Linearity of Quantitative Measurement Procedures: A Statistical Approach. Samples were

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prepared by diluting a serum sample with a high 25-OH vitamin D concentration with a serum sample with a low 25-OH vitamin D concentration to obtain eleven (11) intermediate concentration levels across the measuring range. Samples were run in four replicates, using one reagent lot. The samples tested ranged from 3.4 to 128.5 ng/mL. The linear regression of the observed mean concentrations versus the expected concentration was calculated.

The resulting linear regression equation was $y = 1.02x - 0.56 \, \text{ng/mL}$, $R^2 = 1.00$

The results of the linearity study support the sponsor's claim that the assay is linear from $4 - 120 \, \text{ng/mL}$.

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

## Standardization and Traceability

The ST AIA-PACK 25-OH Vitamin D Assay was originally cleared under $510(\mathrm{k})$ k123131. Tosoh has modified the assay by standardizing the cleared vitamin D assay in accordance with the Vitamin D Standardization Program (VDSP). The VDSP is an international collaborative effort to standardize the laboratory measurement of serum 25-OH vitamin D. This collaboration involves the coordinated efforts of the National Institutes of Health, Office of Dietary Supplements (ODS), the Centers for Disease Control and Prevention (CDC), the National Institutes for Standards and Technology (NIST), Ghent University, and other institutions. Please refer to http://ods.od.nih.gov/Research/VitaminD.aspx for more information on the VDSP program.

To achieve standardization against the VDSP recognized Reference Measurement Procedure (RMP), the ST AIA-PACK 25-OH Vitamin D Assay master calibration parameters were aligned to the CDC VDSP by using 107 human serum samples from the CDC VSDP program which were value assigned by the RMP, Ghent University ID-LC-MS/MS and traceable to NIST SRM2972.

## Calibrator traceability and value assignment

This assay is traceable to the isotope dilution-liquid chromatography/tandem mass spectrometry (ID-LC-/MS/MS) 25-OH Vitamin D Reference Method Procedure (RMP) that is traceable to the National Institute of Standards and Technology (NIST) Standard Reference Material (SRM) 2972. The calibrator traceability and value assignment are prepared according to an internal procedure.

The values of the ST AIA-PACK 25-OH Vitamin D primary reference materials were assigned to align with CDC VDSP recognized RMP, Ghent University ID-LC-MS/MS. The values of the secondary reference material were assigned using the AIA instruments with the primary reference materials as calibrators. The values of the ST AIA-PACK 25-OH Vitamin D Calibrators supplied to the user, are assigned using the AIA instruments with the secondary reference materials as calibrators. The ST AIA-PACK 25-OH Vitamin D Calibrator target values are listed below:

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Calibrator Level 1: 0 ng/mL
Calibrator Level 2: 8 ng/mL
Calibrator Level 3: 17 ng/mL
Calibrator Level 4: 33 ng/mL
Calibrator Level 5: 66 ng/mL
Calibrator Level 6: 135 ng/mL

The ST AIA-PACK 25-OH Vitamin D assay (candidate device) has passed the certification process for the CDC VDSP and a certificate has been provided by the CDC. Please see http://www.cdc.gov/labstandards/hs.html for more information on the CDC VDSP certification program.

## Stability

The calibrator shelf-life and open-vial stability testing protocols and acceptance criteria were previously reviewed in k123131. Reconstituted calibrators are stable for one day when stored at 2-8° C.

## 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), three blank samples (human serum based calibrator 1) were assayed 60 times using two reagent lots over multiple days on two analyzers. The Limit of Blank was the value at the 95th percentile and was determined to be 1.6 ng/mL.

To determine the Limit of Detection (LoD), four low level human serum samples, with concentrations in the range from LoB to approximately 4xLoB, were measured in four replicates per day for three days on two analyzers using two reagent lots for a total of 48 measurements per sample. The LoD was determined to be 3.2 ng/mL using the following equation: LoD=LoB + C_B X SD.

To determine the Limit of Quantification (LoQ), four low level human serum samples, with concentrations in ranging from LoB to approximately 5xLoB, were measured in replicates of 8 for five days on one analyzer using one reagent lot for a total of 40 measurements per sample. The LoQ claim is 3.3 ng/mL based on a functional sensitivity at 20% CV.

Detection limits results:

| Limit of Blank | Limit of Detection | Limit of Quantitation |
| --- | --- | --- |
| 1.6 ng/mL | 3.2 ng/mL | 3.3 ng/mL |

The claimed measuring range of the assay is 4-120 ng/mL.

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e. Analytical specificity:

Potential interference from common endogenous substances, as well as potential cross reactivity of substances with a similar chemical structure as 25(OH) vitamin D, was evaluated in k123131.

The package insert contains the following limitation: Do not use hemolyzed samples. Hemolyzed samples will give erroneous results.

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 (standardized ST AIA-PACK 25-OH Vitamin D assay) to the predicate device (non-standardized ST AIA-PACK 25-OH Vitamin D assay, k123131). A total of 181 native serum samples were tested in singleton on an AIA-200 analyzer. Deming regression analysis was performed and the results are shown below:

Standardized vs non-standardized ST AIA-PACK 25-OH Vitamin D

| N=181 | Deming regression (95%CI) |
| --- | --- |
| Slope | 0.808 (0.801 to 0.816) |
| Intercept | 0.26 (-0.15 to 0.67) |
| r | 0.9979 |
| Range tested | 9.4 to 92.3 ng/mL |

The slope showed an approximate bias of $-20\%$ between the candidate device and the predicate device; however, this shift is expected because the purpose of the device modification was to adjust the calibration to better align with the VDSP reference sample concentration target levels. Therefore, test results from the candidate device do not, and are not expected to, directly correlate with test results from the predicate device. Laboratories that use the Tosoh ST AIA-PACK 25-OH Vitamin D should be aware of the significantly different performance of the modified assay.

An additional method comparison study was conducted to evaluate the accuracy between the candidate device and the RMP, University of Ghent's ID-LC/MS/MS method. The method comparison against the RMP was the basis of the substantial equivalence determination. In this method comparison study, a total of 111 unaltered serum samples value assigned by ID-LC-MS/MS RMP (Ghent University) were tested in singleton on an AIA-200 analyzer using the candidate device. Results from the Weighted Deming regression analysis is shown below:

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ST-AIA PACK 25-OH Vitamin D vs Ghent ID-LC-MS/MS RMP

| N=111 | Weighted Deming regression (95% CI) |
| --- | --- |
| Slope | 0.98 ( 0.92 to 1.05) |
| Intercept | -0.48 ( -1.89 to 0.92 ) |
| r | 0.965 |
| Range tested | 4.7 to 120 ng/mL |

b. Matrix comparison:

Matrix comparison was evaluated in k123131.

3. Clinical studies:

a. Clinical Sensitivity:

Not applicable

b. Clinical specificity:

Not applicable

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

Not applicable

4. Clinical cut-off:

Not applicable

5. Expected values/Reference range:

The reference range study was conducted with reference to the CLSI C28-A3 guideline. A total of 252 serum specimens from 111 females and 141 males from ages 21 to 86 years were assayed in singleton utilizing the ST AIA-PACK 25-OH Vitamin D assay on the AIA-2000 analyzer. The specimens were obtained from the diverse regions of the United States to represent a broad spectrum of UV light exposure in the intended use population. The samples were obtained from apparently healthy light skin and dark skin individuals who were not taking Vitamin D supplements of more than 2000 IU. Specimens with abnormal PTH, TSH, Calcium, Magnesium and Phosphorus were excluded from the data. The 95% reference interval was calculated by a non-parametric method and the following range was obtained:

Normal Adults: 12.3 ng/mL to 60.0 ng/mL (n=252)

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N. Proposed Labeling:

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

O. Conclusion:

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

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