The ADVIA Centaur Vitamin D Total (VitD) assay is for in vitro diagnostic use in the quantitative determination of total 25 (OH) vitamin D in human serum and plasma (EDTA, lithium heparin, sodium heparin) using the ADVIA Centaur XP system. The ADVIA Centaur VitD assay is intended as an aid in the determination of vitamin D sufficiency.

The ADVIA Centaur Vitamin D Total (VitD) Calibrators are for in vitro diagnostic use in calibrating ADVIA Centaur systems Vitamin D Total (VitD) assay.

The ADVIA Centaur Vitamin D Total (VitD) OC is for in vitro diagnostic use to monitor the precision and accuracy of the ADVIA Centaur VitD assay on the ADVIA Centaur systems.

The ADVIA Centaur Vitamin D Total (VitD) Master Curve Material is for in vitro diagnostic use in the verification of calibration and reportable range of the ADVIA Centaur VitD assay.

Device Description

The ADVIA Centaur VitD reagent kit comes in two configurations (100 or 500 test kit) and each kit consists of the following:

ReadyPack primary reagent:
Lite Reagent: 5.0 mL/reagent pack: anti-VitD (monoclonal mouse) antibody labeled with acridinium ester (~0.8 µg/mL) in buffer with bovine serum albumin, mouse IgG, and sodium azide (<0.1%).

Solid Phase: 10.0 mL/reagent pack: anti-fluorescein (monoclonal mouse)-coated paramagnetic particles (PMP) (~0.60 mg/mL) in buffer with bovine serum, albumin, surfactant, and sodium azide (<0.1%).

Ancillary Well Reagent: 5.0 mL/reagent pack: vitamin D-analog conjugated to fluorescein (~0.2 ug/mL) and 1-anilinoaphthatlene-8-sulfonic acid in buffer with bovine serum albumin and sodium azide (<0.1%).

The ADVIA Centaur Vitamin D Total calibrators are lyophilized human plasma with 2 levels of 25 (OH) vitamin D concentrations. After reconstitution, calibrators contain low or high levels of 25 (OH) vitamin D in buffered, defibrinated human plasma with bovine serum albumin, cholesterol, preservatives, and sodium azide (< 0.1%).
The ADVIA Centaur Vitamin D Total QC and The ADVIA Centaur Master Curve Materials (MCM) are sold separately and are described below.
The ADVIA Centaur Vitamin D Total QC (sold separately) are lyophilized human plasma with 2 levels of 25 (OH) vitamin D concentrations. After reconstitution, calibrators contain low or high levels of 25 (OH) vitamin D in buffered, defibrinated human plasma with bovine serum albumin, cholesterol, preservatives, and sodium azide (< 0.1%).
The ADVIA Centaur Master Curve Materials (MCM) (sold separately) are lyophilized bovine serum albumin with 5 levels of 25 (OH) vitamin D concentrations. After reconstitution, materials contain different levels of 25 (OH) vitamin D in buffered, defibrinated human plasma with bovine serum albumin, cholesterol, preservatives, and sodium azide (< 0.1%).

The ADVIA Centaur Vitamin D Total (VitD) calibrators, controls and MCMs were prepared from human blood components tested using FDA approved methods and shown to be negative for hepatitis B surface antigen, anti-hepatitis C and anti-HIV 1 and 2 antibodies.

The ADVIA Centaur VitD calibrators, controls, and master curve verifier have been previously cleared in K110586. There are no reagent or material changes for the calibrators, controls, and master curve verifier, just a change in the traceability. The calibrators are no longer sold in separate package but sold together with the reagent kit.

AI/ML Overview

Here's an analysis of the provided text to extract the requested information, focusing on the study related to the modification of the ADVIA Centaur Vitamin D Total (VitD) Assay, as this is the primary focus of the 510(k) submission. The previous clearance (K110586) is referenced for other performance characteristics not directly part of the modification.

1. Table of Acceptance Criteria and Reported Device Performance

The submission primarily focuses on aligning the device's traceability and calibration to the Vitamin D Standardization Program (VDSP). The key acceptance criteria revolve around achieving this alignment and maintaining analytical performance characteristics.

Acceptance Criteria Category	Specific Criteria (Implicit/Explicit)	Reported Device Performance
Traceability/Standardization	Alignment with VDSP recognized Reference Measurement Procedure (RMP) (University of Ghent's ID-LC/MS/MS 25 (OH) vitamin D RMP)	Deming regression: y=0.99x + 0.53 ng/mL, r=0.96 (Comparing ADVIA Centaur VitD Assay to University of Ghent's ID-LC/MS/MS)
	Certification by CDC VDSP	Passed CDC VDSP certification
Method Comparison (vs. RMP)	Good correlation with RMP, especially around medical decision ranges (10-30 ng/mL)	Deming regression: y=0.93x + 2.89, r=0.99 (Comparing candidate device to RMP). "Correlate well around medical decision range of 10 to 30 ng/mL; however results above 50 ng/mL starts to show some scatter. However, the scatter in that part of the assay range is clinically insignificant."
Precision/Reproducibility	Acceptable %CV values for within-run and total imprecision	%CV for within-run ranged from 3.0% to 5.3%. %CV for total ranged from 4.2% to 11.9%. (Meeting CLSI Guideline EP5-A2 implicitly)
Linearity/Reportable Range	Demonstrates linearity across the claimed measuring range (4.2-150 ng/mL) with good recovery.	y=0.99x + 0.04, r=0.999. % Recovery for samples across the range of 2.92-174.11 ng/mL was between 94% and 108%. Claimed measuring range 4.2-150 ng/mL.
Detection Limit (LoB, LoD, LoQ)	Determined according to CLSI guidelines EP17-A2	LoB: 1.7 ng/mL, LoD: 3.2 ng/mL, LoQ: 4.2 ng/mL
Analytical Specificity (Interference)	Identify and characterize potential interferents (e.g., Fluorescein) and specify limitations.	Fluorescein interference detected. At 0.10 mcg/mL, interference caused 8% and 7% bias for two different vitamin D concentrations, leading to a limitation statement: "Do not use samples which contain fluorescein levels > 0.10 ug/mL can produce falsely elevated results in this assay."
Stability (Shelf Life, Open Vial)	Maintain analytical performance over specified storage conditions.	Shelf life: 26 weeks (6 months) at 2-8º C. Open vial: 120 days at -20° C, 28 days at 2-8° C, 7 days at 25° C.
Expected Values/Reference Range (Pediatric)	Establish a pediatric reference range following CLSI guidelines.	Observed Range (2.5th to 97.5th percentile): 11.4 ng/mL to 46.0 ng/mL for pediatric patients (1-21 years).

2. Sample Sizes Used for the Test Set and Data Provenance

Standardization/Traceability Study:
- Sample Size: 170 unique serum samples.
- Data Provenance: Not explicitly stated, but the reference to "University of Ghent's ID-LC/MS/MS 25 (OH) vitamin D Reference Measurement Procedure (RMP)" suggests international collaboration. These are clinical samples used for method correlation.
Method Comparison Study (Candidate Device vs. Predicate Device):
- Sample Size: 177 native human serum samples.
- Data Provenance: Not explicitly stated. Assumed to be clinical samples.
Method Comparison Study (Candidate Device vs. RMP - University of Ghent):
- Sample Size: 122 independent serum samples (116 native and 6 spiked samples).
- Data Provenance: Clinical samples.
Precision/Reproducibility:
- Sample Size: 2 natural patient serum samples and 4 Medical Decision Pools (MDP). Each tested 80 replicates total.
- Data Provenance: Not explicitly stated, but refers to "natural patient serum samples" and "human serum sample pools."
Linearity/Assay Reportable Range:
- Sample Size: 1 individual patient serum sample (spiked) and 1 low sample (acid treated human serum, charcoal treated, spiked). These were used to generate 9 dilutions.
- Data Provenance: Human serum.
Detection Limit (LoB, LoD, LoQ):
- Sample Size: 5 treated human serum samples for LoB, 6 low serum samples for LoD, 6 low human samples for LoQ.
- Data Provenance: Human serum.
Analytical Specificity (Fluorescein Interference):
- Sample Size: 1 serum sample (spiked) for initial screening, 2 serum pools (spiked) for dose response.
- Data Provenance: Human serum.
Expected Values/Reference Range (Adults):
- Sample Size: 291 apparently healthy adults.
- Data Provenance: North and South of the U.S., collected during different seasons (summer and winter).
Expected Values/Reference Range (Pediatric):
- Sample Size: 237 pediatric patients.
- Data Provenance: North and South of the U.S., collected during different seasons (summer and winter).

All studies appear to be prospective in nature, as they involve testing samples specifically for these evaluations.

3. Number of Experts Used to Establish the Ground Truth for the Test Set and Their Qualifications

For the calibration and method comparison studies directly related to the modification, the "ground truth" (or reference standard) was established by the University of Ghent's ID-LC/MS/MS 25 (OH) vitamin D Reference Measurement Procedure (RMP). This is a highly specialized analytical method, not typically performed by "experts" in the sense of medical professionals diagnosing patients, but rather by highly qualified analytical chemists/scientists with expertise in mass spectrometry and vitamin D analysis. The document states it's "traceable to the National Institute of Standards and Technology Standard Reference Material 2972," indicating a gold standard laboratory method. The CDC VDSP also provided certification, implying their expert oversight of the standardization process.

For establishing adult and pediatric reference ranges, the ground truth was based on samples from "apparently healthy" individuals whose PTH, TSH, and calcium values were normal (adults) and who met stringent inclusion/exclusion criteria (pediatrics). The definition of "healthy" and the clinical assessment for inclusion would have implicitly involved medical professionals, but their specific number or qualifications are not detailed beyond the rigorous selection criteria.

4. Adjudication Method for the Test Set

This type of immunoassay (quantitative chemiluminescent) does not typically involve expert adjudication in the way, for example, imaging AI algorithms do. The "ground truth" for the standardization and method comparison studies is the measured value from the RMP (University of Ghent's ID-LC/MS/MS). For precision, linearity, and detection limit studies, the results are statistical derivations from repeated measurements. For interference, it's based on a calculated bias. Therefore, no multi-expert adjudication method (like 2+1 or 3+1) is relevant or described.

5. If a multi-reader multi-case (MRMC) comparative effectiveness study was done, If so, what was the effect size of how much human readers improve with AI vs without AI assistance

No MRMC study was done. This device is a laboratory assay (in vitro diagnostic device) that directly measures an analyte concentration. It is not an AI-powered imaging or diagnostic tool that assists human readers.

6. If a standalone (i.e., algorithm only without human-in-the-loop performance) was done

Yes, the studies reported are for the standalone performance of the assay system (ADVIA Centaur XP system with the VitD assay). The device directly measures 25(OH) vitamin D concentration from patient samples. There is no human-in-the-loop component for the measurement itself; human operators perform the testing and interpret the numerical results.

7. The type of ground truth used

The primary ground truth used for the device modification and its substantial equivalence claim is Reference Measurement Procedure (RMP) values from the University of Ghent's ID-LC/MS/MS 25 (OH) vitamin D method, which is traceable to NIST SRM 2972. This is a highly accurate and precise analytical method considered the gold standard for vitamin D measurement.

For establishing reference ranges, the ground truth was indirectly informed by the physiological status of "apparently healthy" individuals as assessed by clinical criteria and other biomarker levels (PTH, TSH, calcium).

8. The sample size for the training set

The document does not explicitly describe a separate "training set" in the context of machine learning. For traditional in vitro diagnostic assays like this, the manufacturer develops and refines the assay based on internal research and development. The "standards" and "calibrators" used in the assay are crucial for its performance. The document states:

"a set of 10 standards have been manufactured whose values have been assigned by method correlation to the RMP [University of Ghent's ID-LC/MS/MS]". This refers to how the internal calibration system was developed or aligned to the RMP.
"Calibrators, 2-level controls...and MCMs are traceable to internal standards, which are traceable to the Ghent University ID-LC-MS/MS."
"Production lots of calibrators, controls and master curve materials (MCM) are value assigned against the internal standards using two reagent lots, 2 runs on two different instruments for a total of 24 replicates."

While not a "training set" in the AI sense, these internal standards and calibrators essentially "train" or set the operating parameters of the assay. The 170 unique serum samples (described in the traceability section) used for the initial method correlation to the RMP served as critical data for aligning the assay to the VDSP.

9. How the ground truth for the training set was established

The "ground truth" for establishing the internal standards and calibrators (which "train" the assay) was established by correlation to University of Ghent's ID-LC/MS/MS 25 (OH) vitamin D Reference Measurement Procedure (RMP). The RMP itself is a highly validated and accurate method for measuring 25(OH) vitamin D, representing the scientific "ground truth" for this measurand. The process involved:

Measuring 170 unique serum samples using the RMP to get assigned values.
Using these RMP-assigned values to correlate and assign values to manufactured internal standards.
Then, using these RMP-traceable internal standards to value-assign the actual calibrators, controls, and MCMs provided with the kit.

Summary

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

A. 510(k) Number:

K133156

B. Purpose for Submission:

Modification of traceability for standardization, and addition of pediatric reference range

C. Measurand:

25-hydroxyvitamin D

D. Type of Test:

Quantitative chemiluminescent

E. Applicant:

Siemens Healthcare Diagnostics

F. Proprietary and Established Names:

ADVIA Centaur Vitamin D Total (VitD) Assay

ADVIA Centaur Vitamin D Total (VitD) Calibrators

ADVIA Centaur Vitamin D Total (VitD) Quality Control (QC)

ADVIA Centaur Vitamin D Total Master (VitD) Curve Material

G. Regulatory Information:

1. Regulation section:
- 21 CFR 862.1825, Vitamin D Test System
- 21 CFR 862.1150, Calibrator
- 21 CFR 862.1660, Quality Control Material (Assayed and Unassayed)
1. Classification:

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Class II

Class I, reserved

1. Product code:
  MRG, Vitamin D Test System

JJX, Single (specified) Analyte Controls

JIT, Calibrator, secondary

1. Panel:
  Clinical Chemistry (75)

H. Intended Use:

1. Intended use(s):
  See Indications for use below
1. Indication(s) for use:
  The ADVIA Centaur Vitamin D Total (VitD) assay is for in vitro diagnostic use in the quantitative determination of total 25 (OH) vitamin D in human serum and plasma (EDTA, lithium heparin, sodium heparin) using the ADVIA Centaur XP system. The ADVIA Centaur VitD assay is intended as an aid in the determination of vitamin D sufficiency.

The ADVIA Centaur Vitamin D Total (VitD) Calibrators are for in vitro diagnostic use in calibrating ADVIA Centaur systems Vitamin D Total (VitD) assay.

The ADVIA Centaur Vitamin D Total (VitD) OC is for in vitro diagnostic use to monitor the precision and accuracy of the ADVIA Centaur VitD assay on the ADVIA Centaur systems.

The ADVIA Centaur Vitamin D Total (VitD) Master Curve Material is for in vitro diagnostic use in the verification of calibration and reportable range of the ADVIA Centaur VitD assay.

1. Special conditions for use statement(s):
  For prescription use only

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4. Special instrument requirements:

For use on the ADVIA Centaur XP instrument

I. Device Description:

The ADVIA Centaur VitD reagent kit comes in two configurations (100 or 500 test kit) and each kit consists of the following:

1. ReadyPack primary reagent:
  Lite Reagent: 5.0 mL/reagent pack: anti-VitD (monoclonal mouse) antibody labeled with acridinium ester (~0.8 µg/mL) in buffer with bovine serum albumin, mouse IgG, and sodium azide (<0.1%).

Solid Phase: 10.0 mL/reagent pack: anti-fluorescein (monoclonal mouse)-coated paramagnetic particles (PMP) (~0.60 mg/mL) in buffer with bovine serum, albumin, surfactant, and sodium azide (<0.1%).

1. The ADVIA Centaur Vitamin D Total calibrators are lyophilized human plasma with 2 levels of 25 (OH) vitamin D concentrations. After reconstitution, calibrators contain low or high levels of 25 (OH) vitamin D in buffered, defibrinated human plasma with bovine serum albumin, cholesterol, preservatives, and sodium azide (< 0.1%).
  The ADVIA Centaur Vitamin D Total QC and The ADVIA Centaur Master Curve Materials (MCM) are sold separately and are described below.
1. The ADVIA Centaur Vitamin D Total QC (sold separately) are lyophilized human plasma with 2 levels of 25 (OH) vitamin D concentrations. After reconstitution, calibrators contain low or high levels of 25 (OH) vitamin D in buffered, defibrinated human plasma with bovine serum albumin, cholesterol, preservatives, and sodium azide (< 0.1%).
1. The ADVIA Centaur Master Curve Materials (MCM) (sold separately) are lyophilized bovine serum albumin with 5 levels of 25 (OH) vitamin D concentrations. After reconstitution, materials contain different levels of 25 (OH) vitamin D in buffered, defibrinated human plasma with bovine serum albumin, cholesterol, preservatives, and sodium azide (< 0.1%).

The ADVIA Centaur VitD calibrators, controls, and master curve verifier have been

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previously cleared in K110586. There are no reagent or material changes for the calibrators, controls, and master curve verifier, just a change in the traceability. The calibrators are no longer sold in separate package but sold together with the reagent kit.

J. Substantial Equivalence Information:

1. Predicate device name(s): ADVIA Centaur Vitamin D Total (VitD) Assay, including calibrators, controls, and master curve verifiers.
1. Predicate 510(k) number(s): K110586
1. Comparison with predicate:

Assay:

Similarities
Item	Candidate DeviceADVIA Centaur VitDAssay (modified)	Predicate DeviceADVIA Centaur VitDAssay (K110586)
Intended Use	For the in vitro diagnosticuse in the quantitativedetermination of total 25(OH) vitamin D in humanserum and plasma (EDTA,lithium heparin, sodiumheparin) using the ADVIACentaur XP system.	Same
Indications for Use	This assay is intended to beused as an aid in thedetermination of vitamin Dsufficiency.	Same
Sample type	Serum and Plasma	Same
Measurement	Quantitative	Same
Assay Principle	Competitive immunoassay	Same
Technology	Chemiluminescence	Same
Detection Antibody	Monoclonal mouse antibodylabeled with acridium ester(AE)	Same
Capture Antibody	Anti-fluorescein labeled(FITC) monoclonal mouseantibody covalently boundto paramagnetic particles(PMP).	Same
Assay Range	4.2-150 mg/mL	Same
Calibration	2 point	Same

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Similarities
Item	Candidate DeviceADVIA Centaur VitDAssay (modified)	Predicate DeviceADVIA Centaur VitDAssay (K110586)
Calibrators	ADVIA Centaur VitDCalibrators	Same

Differences
Item	Candidate DeviceADVIA Centaur VitD Assay(modified)	Predicate DeviceADVIA Centaur VitDAssay (K110586)
Standardization/Traceability	Traceable to the ID-LC/MS/MS 25 (OH) vitaminD Reference MeasurementProcedure(University of Ghent) viapatient sample correlation	Traceable to an internalLC-MS/MS via patientsample correlation
Calibrators packaging	Provided with reagent kit	Provided separately
Expected Values (pediatric)	Pediatric:11.4 to 45.8 ng/mL	Pediatric: not provided
Expected Values (adult)	Adult: 7.4 to 44.0 ng/mL	Adult: 10.6-43.4 ng/mL

Controls:

Similarities
Item	Candidate DeviceADVIA Centaur VitDControls (modified)	Predicate DeviceADVIA Centaur VitDControls (K110586)
Intended Use	For the in vitro diagnosticuse to monitor the precisionand accuracy of the ADVIACentaur VitD assay on theADVIA Centaur systems.	Same
Antigen	25 (OH) vitamin D3	Same
Number of levels	2	Same
Matrix	lyophilized human plasma	Same
Packaging	Provided separately	Same
Storage temperature	2-8°C	Same

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Differences
Item	Candidate DeviceADVIA Centaur VitDControls (modified)	Predicate DeviceADVIA Centaur VitDControls (K110586)
Standardization/Traceability	Traceable to the ID-LC/MS/MS 25 (OH) vitaminD Reference MeasurementProcedure(University of Ghent) viapatient sample correlation	Traceable to an internalLC-MS/MS via patientsample correlation

Master curve materials (MCM):

Similarities
Item	Candidate Device	Predicate Device
	ADVIA Centaur VitDMCM (modified)	ADVIA Centaur VitDMCM (K110586)
Intended Use	For in vitro diagnostic usein the verification ofcalibration and reportablerange of the ADVIACentaur VitD assay	Same
Antigen	25 (OH) vitamin D	Same
Number of levels	5	Same
Matrix	Lyophilized human plasma	Same
Packaging	Provided separately	Same
Storage temperature	2-8°C	Same

Differences
Item	Candidate DeviceADVIA Centaur VitD MCM(modified)	Predicate deviceADVIA Centaur VitD MCM (K110586)
Standardization/Traceability	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 LC-MS/MS via patient sample correlation

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

CLSI Guideline EP5-A2: Evaluation of Precision Performance of Qualitative Measurement Methods

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CLSI Guideline EP17-A: Protocols for Determination of Limits of Detection and Limits of Quantitation.

CLSI Guideline EP6-A: Evaluation of the Linearity of Qualitative Measurement Methods

CLSI Guideline C28-A2: How to Define and Determine Reference Intervals in the Clinical Laboratory: Approved Guideline

CLSI Guideline EP7-A2: Interference Testing in Clinical Chemistry; Approved Guideline

L. Test Principle:

The ADVIA Centaur Vitamin D Total assay is a one-pass 18 minute competitive immunoassay that uses an anti-fluorescein labeled (FITC) monoclonal antibody covalently bound to paramagnetic particles (PMP), one monoclonal antibody labeled with acridium ester (AE), and a vitamin D analog labeled with fluorescein. An inverse relationship exists between the amount of vitamin D present in the patient sample and the amount of relative light units (RLUs) detected by the system.

M. Performance Characteristics (if/when applicable):

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

Within run and total imprecision were evaluated by testing two natural patient serum samples and four Medical Decision Pools (MDP) which consisted of 25 (OH) vitamin D spiked into human serum sample pools. Each sample was assayed in 2 replicates per run, 2 runs per day for 20 days for a total of 80 replicates. Testing was performed with two reagent lots on two analyzers. The precision results from a representative reagent lot are summarized below:

		Within Run			Total
Sample	N	Mean(ng/mL)	SD	%CV	SD	%CV
Patient 1	80	13.6	0.64	4.7	1.61	11.9
Patient 2	80	17.2	0.91	5.3	1.70	9.9
MDP pool 1	80	28.2	1.45	5.2	2.02	7.2
MDP pool 2	80	46.1	1.79	3.9	2.79	6.1
MDP pool 3	80	73.2	2.71	3.7	4.36	6.0
MDP pool 4	80	114.1	3.44	3.0	4.71	4.2

b. Linearity/assay reportable range:

Dilutions were prepared using an individual patient serum sample with a 25 (OH)

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vitamin D value of 22 ng/mL spiked with 25 (OH) vitamin D3 to obtain a 25 (OH) vitamin D total value of approximately 180 ng/mL, and a low sample which was prepared by acid treating human serum followed by the addition of charcoal and centrifugation then spiked with low level 25(OH) vitamin D3 near the LoD of the assay. The high serum sample and the low sample were combined in different ratios to produce 9 dilutions covering the assay range. The dilutions were run in replicates of 3 on a single analyzer using 1 lot of reagent. Linearity was evaluated by calculating a linear regression comparing observed values versus expected values. Results of the percent recovery of all the samples are summarized below:

Sample	Expectedconcentration(ng/mL)	Observedconcentration(ng/mL)	%Recovery
P1	2.92	2.92	100
P2	24.32	26.2	108
P3	45.72	42.83	94
P4	67.12	65.92	98
P5	88.52	87.06	98
P6	109.91	112.88	103
P7	131.31	124.19	95
P8	152.71	149.58	98
P9	174.11	174.11	100

A weighted least squares linear regression analysis resulted in the following equation:

y=0.99x + 0.04 r=0.999

The sponsor claims a measuring range of 4.2-150 ng/mL for the ADVIA Centaur Vitamin D assay.

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

Standardization and Traceability

The Siemens ADVIA Centaur Vitamin D Total was originally cleared under 510(k) K110586. Siemens 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.

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To achieve standardization against the VDSP recognized Reference Measurement Procedure (RMP), one hundred and seventy unique serum samples were obtained with 25 (OH) vitamin D values assigned using the University of Ghent's ID-LC/MS/MS 25 (OH) vitamin D Reference Measurement Procedure (RMP). The values ranged from 5 to 100 ng/mL. Using these samples, a set of 10 standards have been manufactured whose values have been assigned by method correlation to the RMP. This value assignment has aligned the candidate device to the RMP. The RMP is traceable to the National Institute of Standards and Technology Standard Reference Material 2972.

The relationship between the ADVIA Centaur VitD Assay and ID-LC/MS/MS 25-OH vitamin D RMP is described using Deming regression:

y=0.99 x + 0.53 ng/mL, r=0.96

The ADVIA Centaur Vitamin D Total assay (candidate device) has passed the certification process for the CDC VDSP and a certificate has been provided by the CDC. Please see the certification process at http://www.cdc.gov/labstandards/hs.html.

Value assignment:

Calibrators, 2-level controls (Level 1: 20 ng/mL and Level 2: 100 ng/ml) and MCMs are traceable to internal standards, which are traceable to the Ghent University ID-LC-MS/MS. The internal standards were value assigned via method correlation using clinical samples with the Ghent University ID-LC/MS/MS assigned values.

Production lots of calibrators, controls and master curve materials (MCM) are value assigned against the internal standards using two reagent lots, 2 runs on two different instruments for a total of 24 replicates. The average dose for each calibrator, control and MCM assigns their value.

Stability:

Shelf life stability studies: Real-time stability studies were performed for the control materials, MCM and the calibrators. The stability study protocol and the acceptance criteria have been reviewed and found to be acceptable. The real time ongoing stability study supports a stability of 26 weeks (6 months) when materials are stored at 2-8º C.

Open vial stability: The stability study protocol and the acceptance criteria to determine open-vial stability of the control materials, calibrators, and the MCMs have been reviewed and found to be acceptable. Controls, calibrators, and MCMs are stable for up to 120 days when stored at -20° C, 28 days when stored at 2-8° C, and up to 7 days stability when stored at 25° C.

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d. Detection limit:

The limit of blank (LoB), limit of detection (LoD), and the limit of quantitation (LoQ) were determined following CLSI Document EP17-A2 guidelines. The LoD is defined as the lowest concentration of 25 (OH) vitamin D that can be detected with 95% probability. LoB was performed using 5 treated human serum samples and LoD was performed using 6 low serum samples. All samples were assayed with 4 replicates per run, 2 runs per day, for over 5 days, over 2 lots of reagent and on 2 analyzers. LoQ was determined using 6 low human samples (25(OH) vitamin D total values 1.4 to 13 ng/mL), over 4 days, 2 runs per day, 4 replicates, using 2 lots of reagent on 1 analyzer for a total of 40 replicates per sample. LoQ was defined as the lowest concentration with an inter-assay precision of ≤20% CV.

The LoB, LoD and LoQ are summarized below:

LoB	LoD	LoQ
1.7 ng/mL	3.2 ng/mL	4.2 ng/mL

The ADVIA vitamin D assay has a measuring range of 4.2 to 150 ng/mL.

e. Analytical specificity:

See K110586

An additional interferent, Fluorescein, was detected since K110586 was cleared.

An interference study was conducted by the sponsor to characterize the interference with sodium fluorescein in two steps. which includes an initial screening step and a dose response confirmation step. At the first step, sodium fluorescein was spiked into a serum sample with a 25(OH) vitamin D concentration of approximately 22 ng/mL at the following levels: 250 mcg/mL, 2.5 mcg/mL, 0.025 mcg/mL and 0.00025 mcg/mL. The samples were tested in triplicate on the ADVIA Centaur XP. The spiked samples were compared to the unspiked samples. Fluorescein interference was seen at fluorescein levels of 250 mcg/mL and 2.5mcg/mL.

To more accurately determine the level of fluorescein that would no longer interfere with the assay, a dose response testing was performed. Sodium fluorescein was spiked into 2 serum pools with a 25(OH) vitamin D concentration of approximately 25 ng/mL and 49 ng/mL at serially diluted levels: 3.13. 1.56, 0.78, 0.39, 0.20, and 0.10 mcg/mL. These samples were tested along with control samples which did not contain fluorescein. The sponsor defined non-significant interference as the level of fluorescein that will result in ≤ 10% bias. The results of the study are summarized in the table below:

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Fluoresceinmcg/mL	25 (OH) Vit DConcentrationng/mL	PercentInterference
0	25.2	NA
3.13	129.3	414%
1.56	67.6	169%
0.78	42.8	70%
0.39	32.2	28%
0.20	28.4	13%
0.10	27.2	8%
0	49.1	NA
3.13	>150	NA
1.56	118.7	142%
0.78	78.9	61%
0.39	60.4	23%
0.20	55.7	14%
0.10	52.7	7%

The sponsor concluded that the study demonstrated that at a sodium fluorescein level of ≥ 0.10 mcg/mL will cause significant interference.

The following limitation statement has been added to the labeling:

"Do not use samples which contain fluorescein levels > 0.10 ug/mL can produce falsely elevated results in this assay. Interference testing with varying levels of sodium fluorescein as high as 3.13 ug/mL at clinically relevant concentrations of 25(OH) vitamin D (~ 25 - 50 ng/mL) can result in 25(OH) vitamin D concentrations to be greater than 150 ng/mL. Evidence suggests that patients undergoing retinal fluorescein angiography can retain amounts of fluorescein in the body for up to 48 to 72 hours post-treatment. In the cases of patients with renal insufficiency, including many diabetics, retention could be much longer. Such samples can produce falsely elevated values when tested with this assay, and should not be tested.

Inloes R, Clark, D and Drobnies A: Interference of fluorescein, used in retinal angiography with certain clinical laboratory tests. Clin Chem 1987, 33:2126-2127.

Assay cut-off: f.
Not applicable

2. Comparison studies:

a. Method comparison with predicate device:

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A method comparison study was performed to compare the modified (standardized) ADVIA Centaur Vitamin D assay (candidate device) to the non-standardized ADVIA Centaur Vitamin D assay (predicate device), one hundred and seventy-seven native human serum samples with 25 (OH) vitamin D values ranging from 5.0 ng/ml to 140.0 ng/mL were assaved in singlicate using one reagent lot of the candidate device and predicate device. Deming regression was used for the regression analysis and the results are summarized below:

y=0.78x + 3.34, r=0.99

The slope showed an approximate bias of -22% 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 ADVIA Centaur Vitamin D assay 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.

A method comparison study was performed to compare the 25-0H vitamin D concentrations of serum samples using the candidate device and the University of Ghent ID-LC/MS/MS 25-OH vitamin D Reference Measurement Procedure (RMP). One hundred and twenty-two independent serum samples, composed of 116 native and 6 spiked samples, with RMP assigned concentrations ranging from 7.8 ng/mL to 148.1 ng/mL were assayed in a singlicate using one lot of the candidate device. Deming regression was used for the regression analysis and results are summarized below:

y= 0.93x + 2.89, r=0.99

Results correlate well around medical decision range of 10 to 30 ng/mL; however results above 50 ng/mL starts to show some scatter. However, the scatter in that part of the assay range is clinically insignificant.

b. Matrix comparison:
See K110586.
1. Clinical studies:
- a. Clinical Sensitivity:

Not applicable

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b. Clinical specificity:
Not applicable
c. Other clinical supportive data (when a. and b. are not applicable):
Not applicable
1. Clinical cut-off:
  Not applicable
1. Expected values/Reference range:
  Expected Values for Adults:

A reference range study was conducted using adult subjects aged between 21 to 93 years. Samples from 291 apparently healthy adults from the North and South of the U.S. were tested. These samples were collected during different seasons (summer and winter). The samples numbers were split evenly between the North and South, summer and winter, and with and without supplements containing vitamin D. Samples were only included in this study if the samples had normal PTH, TSH, and calcium values. Based on the 95% confidence interval, the following values were established following CLSI guideline C28-A2.

Median 25(OH) vitamin D	Observed Range (2.5th to 97.5th percentile)
22.5 ng/L	7.4 to 44.0 ng/mL

Expected Values for Pediatric patients:

A reference range study was conducted using samples collected from 237 pediatric patients aged between 1 year and up to 21 years. These samples were collected from apparently healthy pediatric subjects from the North and South of the U.S. as well as from different seasons (summer and winter). Serum samples from pediatric patients meeting the following criteria were included:

1. Subjects aged 1 year up to 21-year-old.
1. No active chronic diseases, with the exception of allergic diseases for which no allergy medication has been taken within the last 7 days preceding sample collection.
1. No active acute disease.
1. No prescribed medications in the 7 days preceding sample collection.
1. No contraceptive medication in the 90 days preceding sample collection.

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1. No over the counter medications in the 7 days preceding sample collection.
1. No suspicion of premature of delayed puberty, based on physical examination.
1. No pregnant female subjects.
1. Ages 1 yr to 2yr: Length, weight, and weight for length all within the 10th and 90th percentiles, based on WHO growth charts.
1. < 2 yr: Weight, height, and BMI all within the 10th percentiles based on CDC growth charts.
1. Serum iPTH, TSH levels within published normal ranges
1. Only subjects taking < 1000 IU/Day of Vitamin D supplement
1. Only subjects with no personal or family history of thyroid disease.
1. Serum, iPTH and TSH, levels with published normal ranges.
1. Specimens with serum hemoglobin ≤ 155 mg/dL.

Based on the 95% confidence interval, the following expected values for pediatric patients were established following CLSI guideline C28-A2.

Median 25(OH) vitamin D	23.4 ng/mL
Observed Range 2.5th to 97.5th percentile	11.4 ng/mL to 46.0 ng/mL

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.

Regulation Number and Section

§ 862.1825 Vitamin D test system.

(a)
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.(b)
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.