The ESA Biosciences Inc. Vitamin D HPLC test is for the quantitative determination of 25-hydroxyvitamin D in human serum or EDTA-plasma to be used in the assessment of vitamin D sufficiency. Assay results should be used in conjunction with other clinical or laboratory data to assist the clinician in making individual patient management decisions in an adult population.

Device Description

The ESA method is a complete kit for measurement of Total 25(OH)D by HPLC with electrochemical (EC) detection. Specific reagents and solid phase extraction columns are included for sample preparation and are employed with user-supplied standard laboratory equipment (centrifuge, test tubes, pipettes, etc.). A 200µL volume of sample (serum or plasma) is mixed with a precipitation reagent, which contains internal standard (IS). The internal standard is a stable vitamin D analogue that is used to correct for variability in extraction recovery and analytical sample volume. After centrifugation, supernatant is poured onto a pre-conditioned SPE column for rapid extraction of 25(OH)D and IS. SPE columns are washed with 2 different reagents and analytes are eluted with a third reagent. The resulting eluent is diluted before analysis. The prepared sample is analyzed with an isocratic HPLC system using an ESA EC detector (Coulochem® III or CoulArray®) equipped with a dual coulometric EC cell. Calibration is accomplished by direct HPLC analysis of authentic standard solutions (i.e. not taken through the extraction step). Analysis requires a specific guard and analytical column, mobile phase and calibration reagents to allow rapid quantitative analysis. A dual EC cell is used with the first, upstream, cell maintained at a specific potential to oxidatively screen possible interfering sample components. The second, downstream cell is maintained at a potential that is optimized for selective 25(OH)D detection. The dual coulometric EC cell is a rugged detector that provides much higher selectivity than commonly used absorbance detectors. This allows the use of lower sample volumes than are typically required with HPLC-UV methods and is less susceptible to interferences. Analytical run time is less than 12 minutes and Total 25(OH)D sample concentration is automatically determined by single-point internal standard quantitation.

AI/ML Overview

Here's an analysis of the provided text, outlining the acceptance criteria and study details for the ESA Biosciences Inc. Vitamin D HPLC Test:

1. Table of Acceptance Criteria and Reported Device Performance

The document does not explicitly state "acceptance criteria" in a dedicated section with pass/fail thresholds. However, it presents performance characteristics that implicitly serve as the criteria for evaluating the device's suitability. The table below lists these performance metrics and the reported results.

Performance Characteristic	Acceptance Criteria (Implied)	Reported Device Performance
Precision (Within-run)	Low %CV for various samples	Sample #1 (Plasma): 2.82% CVSample #2 (Plasma): 2.31% CVSample #3 (Serum): 2.55% CVSample #4 (Serum): 2.16% CVLevel 1: 10.6% CVLevel 2: 6.8% CV
Precision (Within-device/Total)	Low %CV for various samples	Sample #1 (Plasma): 6.05% CVSample #2 (Plasma): 5.48% CVSample #3 (Serum): 5.66% CVSample #4 (Serum): 5.43% CVLevel 1: 12.6% CVLevel 2: 8.4% CV
Limit of Detection (LoD)	Defined as concentration with ≤ 5% false negative/positive	5.0 ng/mL
Limit of Quantitation (LoQ)	% CV ≤ 20% at this concentration	7.0 ng/mL
Limit of Blank (LoB)	N/A (reported as a baseline metric)	2.5 ng/mL
Linearity Range	Linear from LoQ to 200 ng/mL with specific bias goals	Linear from 7.0 ng/mL (LoQ) to 200 ng/mL (bias goals: ≤ 3ng/mL bias for ≤ 20 ng/mL; ≤ 7.5% bias for 21-200 ng/mL)
Recovery	Expected % recovery around 100% for spiked samples	Plasma: 90-107% mean recoverySerum: 93-105% mean recovery
Method Comparison (Correlation)	Correlation Coefficient (R) for comparison with reference method (implies r needs to be within an acceptable range, though r < 0.975 was noted as failing an "adequate range test")	R = 0.973
Method Comparison (Bias)	Bias within acceptable limits at different concentrations, especially if correlation is not ideal	At 22.9 ng/mL (LCUV): -2.6 ng/mL (-11.2% bias)At 45.4 ng/mL (LCUV): -5.6 ng/mL (-12.3% bias)At 100.3 ng/mL (LCUV): -3.3 ng/mL (-3.3% bias)
Interference (Cholesterol)	Bias < 10% for cholesterol concentrations < 375 mg/dL	10% positive bias at 500 mg/dL cholesterol; <10% bias for < 375mg/dL
Interference (25(OH)epiD3)	Not to be used in populations where significant interference is expected (e.g., neonates)	Interferes; assay not for use in patients < 1 year of age
Other Interferences	No interference from tested substances at high concentrations	Triglycerides, Albumin, Bilirubin, Hemoglobin, Paricalcitrol: No interference

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

Precision:
- "Neat" Samples: 78-80 replicates per sample (4 samples total), run over 20 days.
- Plasma Pools: 100 replicates per level (2 levels total), run over 20 days.
Limit of Detection (LoD): 133 determinations (67 blank, 66 low-level samples).
Linearity: 13 equally-spaced concentrations, 3 aliquots of each.
Recovery: 5 EDTA plasma subjects, 6 serum subjects; each spiked to 7 concentration levels (0, 5, 10, 20, 50, 100, 200 ng/mL), with 3 replicates each.
Method Comparison: 100 individual patient serum samples (85 from UWHC, 15 augmented samples from a single serum pool).
Interference Studies: High and low (normal) concentrations of each interferent mixed proportionally to produce 5 equally-spaced levels, with 3 replicates each.
Reference Range: 150 apparently healthy adult human subjects.

Data Provenance:

Precision: Not explicitly stated regarding geographic origin, but study followed CLSI EP5-A.
Limit of Detection (LoD): Not explicitly stated regarding geographic origin, but used patient samples and followed CLSI EP17A.
Linearity: Not explicitly stated regarding geographic origin, but followed CLSI EP6.
Recovery: Not explicitly stated regarding geographic origin.
Method Comparison: Serum samples from 85 individual patients collected at the University of Wisconsin Hospitals and Clinics (UWHC). The remaining 15 were augmented from a single serum pool.
Interference Studies: Not explicitly stated regarding geographic origin, but followed CLSI EP7-A.
Reference Range: Samples from 150 apparently healthy adult human subjects collected from 3 locations to achieve even North-South geographical distribution within the latitudes of the contiguous 48 US States. All data appears to be retrospective clinical measurements or prospectively collected for validation purposes (e.g., reference range study).

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

The document does not mention the use of "experts" in the traditional sense of clinicians or radiologists for establishing a ground truth for the test set. Instead, the ground truth for performance characteristics is established through:

Reference Methods: For method comparison, an LCUV reference method was used. The qualifications of the individuals performing the LCUV method or confirming its accuracy are not detailed.
Spiking/Augmenting Samples: For recovery and linearity, known concentrations were added to samples.
Statistical Analysis: CLSI guidelines (EP5-A, EP17A, EP6, EP7-A, EP9-A2, C28-A2) were referenced, implying statistical methods were used to derive performance metrics from direct measurements.

4. Adjudication Method for the Test Set

No adjudication method (e.g., 2+1, 3+1, none) is mentioned as the ground truth was established by reference methods, spiking, or statistical analysis of direct measurements, not by expert consensus on qualitative assessments.

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 performed. The device is a diagnostic test kit for quantitative measurement of vitamin D, not an AI-assisted diagnostic imaging or qualitative assessment tool that involves human readers in that capacity.

6. If a Standalone (i.e., algorithm only without human-in-the-loop performance) Was Done

Yes, the studies reported are standalone performance evaluations of the assay itself. The results are quantitative measurements from the HPLC system, operating without direct human interpretation of the primary data to establish the diagnostic outcome. Human involvement is in sample preparation, running the instrument, and interpreting the numerical output.

7. The Type of Ground Truth Used (expert consensus, pathology, outcomes data, etc.)

The ground truth used for various performance characteristics includes:

Reference Method Measurements: For method comparison, an LCUV reference method served as the ground truth.
Known Concentrations: For linearity and recovery studies, the "ground truth" was the precisely known concentrations of 25(OH)D added to samples.
Statistical Definitions/Calculations: For LoD, LoQ, and precision, the ground truth is derived from statistical analysis of repeated measurements and blank samples according to established CLSI guidelines.
Absence/Presence of Interferents: For interference studies, the ground truth was the known composition and concentration of tested substances in the samples.

8. The Sample Size for the Training Set

The document does not describe a "training set" in the context of machine learning or AI. The development of this HPLC test likely involved extensive method development and optimization, but the reported studies are primarily validation and verification of the finalized method's performance.

9. How the Ground Truth for the Training Set Was Established

As there's no mention of a "training set" in an AI/ML context, this question is not applicable. For the development of the HPLC method, numerous experiments and analytical techniques would have been used to establish the correct parameters and ensure accurate and precise measurement of 25-hydroxyvitamin D. However, these are not described as a formal "training set" with ground truth in the document.

Summary

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Ko7253 6

MAY - 7 2008

510(k) SUMMARY

This summary of 510(k) Safety and effectiveness information is being submitted in
accordance with the requirements of SMDA 1990 and 21 CFR 807.92.

The assigned 510(k) number is:

Submitter:	ESA Biosciences Inc.22 Alpha RoadChelmsford, MA 01824USAPhone: 978-250-7000Fax: 978-250-7090
Contact Person:	Harold AspQuality Assurance Manager
Date of SummaryPreparation:	May 05, 2008
Device Name:	ESA Biosciences Inc. Vitamin D HPLC Test
Classification Name:	Vitamin D Test System 21CFR 862.1825 Product Code:MRG
Predicate Device:	LIASON® 25 OH Vitamin D Assay510(k) 032844
Statement ofIntended Use:	The ESA Biosciences Inc. Vitamin D HPLC test is for thequantitative determination of 25-hydroxyvitamin D inhuman serum or EDTA-plasma to be used in theassessment of vitamin D sufficiency. Assay resultsshould be used in conjunction with other clinical orlaboratory data to assist the clinician in making individualpatient management decisions in an adult population.

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Device Description

The internal standard is a stable vitamin D analogue that is used to correct for variability in extraction recovery and analytical sample volume. After centrifugation, supernatant is poured onto a pre-conditioned SPE column for rapid extraction of 25(OH)D and IS. SPE columns are washed with 2 different reagents and analytes are eluted with a third reagent. The resulting eluent is diluted before analysis. The prepared sample is analyzed with an isocratic HPLC system using an ESA EC detector (Coulochem® III or CoulArray®) equipped with a dual coulometric EC cell. Calibration is accomplished by direct HPLC analysis of authentic standard solutions (i.e. not taken through the extraction step). Analysis requires a specific guard and analytical column, mobile phase and calibration reagents to allow rapid quantitative analysis.

A dual EC cell is used with the first, upstream, cell maintained at a specific potential to oxidatively screen possible interfering sample components. The second, downstream cell is maintained at a potential that is optimized for selective 25(OH)D detection. The dual coulometric EC cell is a rugged detector that provides much higher selectivity than commonly used absorbance detectors. This allows the use of lower sample volumes than are typically required with HPLC-UV methods and is less susceptible to interferences. Analytical run time is less than 12 minutes and Total 25(OH)D sample concentration is automatically determined by single-point internal standard quantitation.

Summary of Performance

Precision

Four 'neat' (i.e., not spiked or pooled) samples consisting of 2 sera and 2 EDTA plasma were used for this study, which followed CLSI EP5-A. Four replicates of each sample, were individually prepared (taken through all pre-analytical steps of the procedure) and analyzed in a single run each day, repeated over 20 days. Samples were run on a single instrument, by two operators and using two lots of reagents and extraction columns. Analysis of variance calculations were used to estimate within-run and within-device imprecision.

Assay	Mean	Within-run		Within-device		N
	ng/mL	SD (ng/mL)	% CV	SD (ng/mL)	% CV
Total 25(OH)D
Sample #1 (Plasma)	22.1	0.63	2.82	1.34	6.05	79
Sample #2 (Plasma)	21.8	0.50	2.31	1.19	5.48	80
Sample #3 (Serum)	25.5	0.65	2.55	1.44	5.66	80
Sample #4 (Serum)	25.6	0.55	2.16	1.39	5.43	78

Precision Performance

Precision performance was also evaluated using plasma pools supplemented with 25(OH)D at 2 concentration levels. The protocol followed CLSI EP5-A. Five replicates of each level were individually prepared (i.e., taken through all pre-analytical steps) and analyzed in a single run each day. Data from a total of 20 runs, performed at two sites were included in the

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precision evaluation (300 total observations). Three different HPLC-EC systems, four operators, two reagent lots, five analytical columns (3 lots), three lots of SPE columns and four analytical cells were used in these studies. Analysis of variance calculations were used to estimate withinrun and total imprecision.

Assay	Mean	Within-run		Total		N
	ng/mL	SD (ng/mL)	% CV	SD (ng/mL)	% CV
Total 25(OH)D
Level 1	43.8	4.6	10.6	5.5	12.6	100
Level 2	117.2	8.0	6.8	9.9	8.4	100

Precision Performance

Limit of Detection (LoD)

The LoD was determined consistent with the guidelines in CLSI EP17A. Blank matrix was bovine serum albumin in phosphate buffered saline. Patient samples with low 25(OH)D concentrations, as determined by the LCUV reference method, were used to prepare low level pools The results were analyzed using the nonparametric method. The LoQ (limit of quantitation) was set to the concentration at which the % CV from low level samples was ≤ 20%.

Provided below are estimates of LoD, limit of blank (LoB) and LoQ based on 133 determinations in 11 runs, with 67 blank and 66 low-level samples with less than 5% false negative and less than 5% false positives:

	ng/mL
Assay	LoB	LoD	LoQ	N
Total 25(OH)D	2.5	5.0	7.0	133

LoD. LoB. and LoQ for Vitamin D

Linearity

Linearity was studied according to CLSI EP6 on two HPLC systems. Low and high level plasma were mixed proportionally to obtain a total of 13 equally -spaced concentrations spanning the intended assay range (7.0 - 200 ng/mL) for 25(OH)D. Three aliquots of each sample concentration were taken through all pre-analytical steps of the assay and analyzed in random order in a single run. Performance was assessed using the polynomial evaluation as described in CLSI EP-6A.

For total 25(OH)D by the ESA Vitamin D HPLC Test, the method has been demonstrated to be linear from the LoQ (7.0 ng/mL) to 200 ng/mL based on goals of ≤ 3ng/mL bias due to nonlinearity for concentrations of ≤ 20 ng/mL and ≤ 7.5% bias due to non-linearity at concentrations of 21-200 ng/mL.

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Recovery

Sample pools: EDTA plasma (5 subjects) and serum (6 subjects) were each spiked with 25HOD concentrate to increase the individual concentrations of 25(OH)D by 0, 5, 10, 20, 50, 100 and 200 ng/mL. Concentration levels of total 25(OH)D in unspiked pools were less than 10 ng/mL. Three replicates of each sample were taken through all pre-analytical steps of the assay. Each pool and corresponding spiked samples were analyzed in random order in a single run. Samples were analyzed on two different HPLC systems.

Percent recovery was determined by: [(Measured concentration - Endogenous (unspiked) concentration)/Spiked Concentration] x 100.

For each sample matrix, mean recovery (n = 6, each level = 3 replicates on 2 systems) are summarized below:

Sample	Spiked Concentration(ng/mL)	% Recovery Mean(n=6)	Standard Deviation% (n=6)
Plasma	10	90	20
	20	100	10
	40	100	11
	100	99	6.8
	200	107	7.2
Serum	10	93	20
	20	94	13
	40	96	8.9
	100	96	5.5
	200	105	3.8

Recovery 25(OH)D

Method Comparison and Bias Estimation

Method comparison studies followed CLSI EP9-A2. The studies were performed at two external sites. Serum samples from 85 individual patients were collected at the University of Wisconsin Hospitals and Clinics (UWHC). These were chosen from samples submitted routinely to UWHC for 25(OH)D analysis and represent a wide population with respect to medical, dietary and therapeutic conditions. Fifteen additional samples were prepared by augmenting aliquots of a single serum pool (prepared from approximately 50 individual patient samples) with varying amounts of 25(OH)D.

All 100 samples were run in duplicate by a LCUV reference method at UWHC. Fifty of these patient samples were run in duplicate by the ESA LCEC test method at one external site and the other fifty patient samples were run in duplicate by the LCEC test method at UWHC. Samples were run on 12 separate days, by 3 different technologists over a 7 week period with no more than 10 patient samples run per day. Calibration and control materials used for the LCUV reference method were independent of the LCEC test method.

For this analysis, data from both sites were pooled and the first replicate of the Y (ESA LCEC) results were analyzed against the average of the X (LCUV) results. No outliers were removed. Five samples with values outside the measuring range, LOQ (7.0 ng/mL) to 200 ng/mL, of the ESA assay were excluded from this analysis.

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The following table and figure provide a summary of data analysis for total 25(OH)D. All data analyses were performed as described in EP9-A2 and include the following:

A Number of points used in the regression and range of X data.
Slope and intercept of fitted ordinary least squares and Deming regression lines. ♪
A From least squares regression: correlation coefficient, standard error of estimate (calculated in the vertical (Y) direction) and confidence intervals for the slope and intercept.
Scatter plots with the line of identity. A
Predicted bias and confidence intervals calculated at various X values from the A partitioned bias method.

25(OH)D
Equation: Least Squares	LCEC = 1.027(LCUV) - 5.3
Slope, 95% Confidence Interval	0.976 to 1.077
Intercept, 95% Confidence Interval (ng/mL)	-8.7 to -1.8
Correlation Coefficient, R	0.973
N	95
Standard Error (ng/mL, y-direction)	9.91
Range (ng/mL)	9.6 – 195
Equation: Deming	LCEC = 1.056(LCUV) - 6.9

Regression Summary -- Total 25(OH)D

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Image /page/5/Figure/0 description: The image is a scatter plot titled "Total 25(OH)D, LCEC vs. LCUV". The x-axis is labeled "LCUV, ng/mL" and ranges from 0 to 200. The y-axis is labeled "LCEC, ng/mL" and ranges from 0 to 200. The plot shows a series of data points labeled "Series 1" and a line labeled "Identity".

Total 25(OH)D - Scatter Plot of First LCEC Replicate vs. Average of Reference Method with Line of Identity

Total 25(OH)D First LCEC Rep vs. Avg. LCUV

Because the correlation coefficient (r) fails the adequate range test (i.e., r < 0.975), the partitioned bias method was used.

	Total 25(OH)D - Bias and Confidence Intervals Using Partitioned Bias Method
--	-----------------------------------------------------------------------------

Average LCUV(ng/mL)	Range ofLCUV(ng/mL)	N	Bias(ng/mL)	Bias(%)	Lower 95%CI Bias*(ng/mL)	Upper 95%CI Bias*(ng/mL)
22.9	9.6 – 34.4	32	-2.6	-11.2	-17.4	-4.9
45.4	34.5 – 56.1	31	-5.6	-12.3	-18.4	-6.1
100.3	57.0 - 195	32	-3.3	-3.3	-8.5	2.0

Confidence intervals calculated at the indicated average LCUV concentration

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Interference Studies

This assay is susceptible to interference from 25(OH)epiD2 and 25(OH)epiD3, which are endogenous metabolites present in neonates . This assay should therefore not be used for measurement in samples from patients < 1 year of age. A 10% positive bias was observed for samples with a cholesterol level of 500mg/dL. Estimated bias for lower concentrations of cholesterol was acceptable (<10%). The following provides additional detail of interference susceptibility testing.

Interference susceptibility testing was performed according to CLSI EP7-A. Substances chosen for study were based on consideration of chemical structure determinants for electrochemical response and chromatographic retention, common medications in the intended population, therapeutic vitamin D analogs and matrix effects / conditions. The following table lists substances that were initially screened by direct HPLC-EC analysis along with the observed retention times.

Compound / Class	Alternate and CommercialNames	Retention time (min.) forpeaks observed onanalytical channel
Lipid-soluble vitamins
Retinyl Acetate		25.06
Retinol		11.5
α-Tocopherol		None
Coenzyme Q10		None
β-Carotene		None
Lutein		None
γ-Tocopherol		None
δ-Tocopherol		None
Lycopene		None
Vitamin D Compounds /Analogues
D3	Cholicalciferol	None
D2	Ergocalciferol	None
1α,25(OH)2D3	Calcitriol, Rocaltrol, Calcijex	4.08
19-nor-1α,25(OH)2D2	Paricalcitol, Zemplar	3.74
Dihydrotachysterol		None
1α,25(OH)2D2		4.7
1α(OH)D3	One-alpha, Alfarol	33.9
1α(OH)D2	Doxercalciferol, Hectorol	34
25(OH)epiD3		8.46

Compounds Analyzed by Direct HPLC Analysis

From Table 9-1, 19-nor-1α,25(OH)-D2 (paricalcitol) and 25(OH)epiD3 were the only compounds that were identified as possible interfering substances. These substances and several endogenous compounds were characterized to determine the degree of interference as a function of interferent concentration (i.e., dose-response). Serum was prepared with high and low (normal) concentrations of each possible interferent (see below). High and low samples were mixed proportionally to produce a total of 5 equally-spaced levels of each possible interfering substance. Three replicates of each sample were individually prepared and analyzed in random order a single run.

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The results of these studies are described below:

The following endogenous substances, when tested according to CLSI EP7-A, in a serum sample containing 25(OH)D2 at 67 ng/mL and 25(OH)D3 at 53 ng/mL, did not interfere at, or below, the interferent test concentration indicated. Bias, calculated from regression analysis of the doseresponse data, exceeding 10% is considered interference.

Substance	High TestConcentration	Comments
Triglycerides	1571 mg/dL	Test Material Triolein
Albumin	12.7 g/dL
Bilirubin	60 mg/dL	.
Hemoglobin	500 mg/dL	Gross hemolysis

The following vitamin D analog, when tested according to CLSI EP7-A, in a serum sample containing 25(OH)D2 at 67 ng/mL and 25(OH)D3 at 53 ng/mL, did not interfere at the interferent test concentration indicated. Bias, calculated from regression analysis of the dose-response data, exceeding 10% is considered interference.

Substance	High TestConcentration	Comments
Paricalcitrol	100 ng/mL	Approx 100 times therapeuticrange for this compound.

The following endogenous substance, when tested according to CLSI EP7-A, in a serum sample containing 25(OH)D2 at 67 ng/mL and 25(OH)D3 at 53 ng/mL was found to produce a positive bias of 10% for both analytes at the high test concentration indicated. Acceptable bias (i.e., < 10%) was found for interferent concentrations < 375mg/dL.

Substance	High TestConcentration
Cholesterol	500 mg/dL

The C3 epimer of 25(OH)D, 25(OH)EpiD3, when tested according to CLSI EP7-A, in a serum sample containing 25(OH)D2 at 67 ng/mL and 25(OH)D3 at 53 ng/mL, was found to interfere with 25(OH)D. As reported by Singh et al.", 25(OH)D2 or D3 epimers may account for a significant proportion (8.7-61.1%) of the total 25(OH)D in infants, with concentrations ranging from 5-92ng/mL. No C3 epimers were detected in age groups greater than 0-1 year. Based on this report, this assay should not be susceptible to interference from C3 epimers for the intended adult patient population.

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Reference Range

The following results were obtained specifically with the ESA Vitamin D HPLC Test from a reference sample group that is reflective of a typical sample population within the contiguous 48 US States. Note: These data are provided as an example. It is important for each laboratory to establish a reference range representative of its typical population.

Samples from 150 apparently healthy adult human subjects were collected from 3 locations to achieve even North-South geographical distribution within the latitudes of the contiguous 48 US States. Samples were collected in late winter. Individuals taking prescription or over-the-counter vitamin D supplements were excluded. The reference range was determined according to the non-parametric method in CLSI C28-A2.

Observed Reference Range and Frequency Distribution

Population (N)	Median 25(OH)D ng/mL	Observed Range ng/mL2.5th to 97.5th Percentile
150	17.4	5.7 - 34.8

Image /page/8/Figure/5 description: The image is a bar graph showing the frequency (%) of a population across different concentration ranges (ng/mL). The x-axis represents the concentration range, while the y-axis represents the frequency percentage. The concentration ranges include <5.0, 5.1-10.0, 10.1-15.0, 15.1-20.0, 20.1-25.0, 25.1-30.0, 30.1-35.0, and 35.1-40.0. The highest frequency is observed in the 15.1-20.0 range, with approximately 25% of the population.

25(OH)D Reference Sample Distribution

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Similarities
Item	Device	Predicate
Intended Use	For the quantitativedetermination of 25-hydroxyvitamin D in humanserum or EDTA-plasma to beused in the assessment ofvitamin D sufficiency. Assayresults should be used inconjunction with other clinicalor laboratory data to assist theclinician in making individualpatient management decisions inan adult population.	For in vitro diagnostic use.Quantitative determination of25-Hydroxyvitamin D (25-OH-D) and other hydroxylatedvitamin D metabolites inhuman serum or plasma to beused in the assessment ofvitamin D sufficiency. Assayresults should be used inconjunction with other clinicallaboratory data to assist theclinician in making patientmanagement decisions in anadult population.
Indications forUse	Determination of Vitamin Dsufficiency.	Same
Matrix	Human serum or plasma	Same
Differences
Item	Device	Predicate
Analyte	25-Hydroxyvitamin D	Same
Methodology	High Performance LiquidChromatography withElectro-Chemical Detection	Chemiluminescent immunoassay
Kit Controls	Recommend commerciallyavailable controls	Two levels, horse serum based,extracted identically to patientsamples
Standards	Working single pointcalibrator is an aqueouscalibration stock solution andinternal stock solution	Stored Master Curve based on 10points, derived from serum basedstandards extracted identically tocontrols and patient samples

Technical Characteristics Compared to Predicate:

Conclusion

Through the use of plasma and serum samples, augmented plasma and serum samples, standards and controls the performance and reliability of this assay has been validated over the measuring range of 7.0 ng/mL (LoQ) to 200 ng/mL. In so doing adequate over the modeling fungs and imits and immits and immunity from interferences has been demonstrated.

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Standards Referenced

EP9-A2 Method Comparison and Bias Estimation Using Patient Samples: Approved 1. Guideline- Second Edition
C28-A2 How to define and Determine Reference Intervals in the Clinical Laboratory; 2. Approved Guideline-Second Addition
EP6-A Evaluation of the Linearity of Quantitative Measurement Procedures: A Statistical 3. Approach; Approved Guideline
EP7-A Interference testing in Clinical Chemistry; Approved Guideline 4.
EP5-A2 Evaluation of Precision Performance of Quantitative Measurement Methods; ട്. Approved Guideline-Second edition

Literature Referenced

Lensmever GL, Wiebe DA, Binkley N, Drezner MK. HPLC method for 25-hydroxyvitamin 1. D measurement: comparison with contemporary assays. Clin Chem 2006;52:1120-6.
1. Singh RJ, Taylor RL, Reddy GS, Grebe SK. C-3 epimers can account for a significant proportion of total circulating 25-hydroxyvitamin D in infants, complicating accurate measurement and interpretation of vitamin D status. J Clin Endocrinol Metab 2006;91:3055-61.

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DEPARTMENT OF HEALTH & HUMAN SERVICES

Image /page/11/Picture/1 description: The image shows the seal for the Department of Health & Human Services - USA. The seal is circular, with the text "DEPARTMENT OF HEALTH & HUMAN SERVICES - USA" arranged around the perimeter of the circle. In the center of the seal is an image of an eagle.

Public Health Service

Food and Drug Administration 2098 Gaither Road Rockville MD 20850

ESA Biosciences Inc. c/o Mr. Harold Asp Quality Assurance Manager 22 Alpha Road Chelmsford, MA 01824

MAY - 7 2008

Re: K072536

Trade/Device Name: Vitamin D HPLC Test Regulation Number: 21 CFR 862.1825 Regulation Name: Vitamin D Test system Regulatory Class: Class II Product Code: MRG Dated: March 24, 2008 Received: March 26, 2008

Dear Mr. Asp:

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.

If your device is classified (see above) into either class II (Special Controls) or class III (PMA), it may be subject to such additional controls. Existing major regulations affecting your device can be found in Title 21, Code of Federal Regulations (CFR), Parts 800 to 895. 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); and good manufacturing practice requirements as set forth in the quality systems (QS) regulation (21 CFR Part 820).

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This letter will allow you to begin marketing your device as described in your Section 510(k) premarket notification. The FDA finding of substantial equivalence of your device to a legally marketed predicate device results in a classification for your device and thus, permits your device to proceed to the market.

If you desire specific information about the application of labeling requirements to your device, or questions on the promotion and advertising of your device, please contact the Office of In Vitro Diagnostic Device Evaluation and Safety at (240) 276-0490. Also, please note the regulation entitled, "Misbranding by reference to premarket notification" (21CFR Part 807.97). You may obtain other general information on your responsibilities under the Act from the Division of Small Manufacturers, International and Consumer Assistance at its toll-free number (800) 638-2041 or (240) 276-3150 or at its Internet address at http://www.fda.gov/cdrh/industry/support/index.html.

Sincerely yours,

Jean M. Cooper, M.S., D.V.M.

Yean M. Cooper, M.S., D.V.M. Director Division of Chemistry and Toxicology Office of In Vitro Diagnostic Device Evaluation and Safety Center for Devices and Radiological Health

Enclosure

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

Indication for Use

510(k) Number (if known):

Device Name: ESA Biosciences Inc. Vitamin D HPLC Test

Indication For Use: K072536

Prescription Use _ X (21 CFR Part 801 Subpart D)

And/Or

Over the Counter Use (21 CFR Part 801 Subpart C)

(PLEASE DO NOT WRITE BELOW THIS LINE; CONTINUE ON ANOTHER PAGE IF NEEDED)

Concurrence of CDRH, Office of In Vitro Diagnostic Device Evaluation and Safety (OIVD)

Di/ Sign. Off.

Division Sign-Off Office of In Vitro Diagnostic Device Evaluation and Safety

510(k) K072536

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.