Predicate Devices

Reference Devices

Not Found

AI/ML (Artificial Intelligence / Machine Learning)

No
The device description and performance studies focus on traditional analytical chemistry techniques (HPLC, EC detection, SPE) and standard statistical methods for validation (CLSI guidelines). There is no mention of AI, ML, or related concepts.

Therapeutic

No
The device is an in vitro diagnostic (IVD) test used to quantitatively determine 25-hydroxyvitamin D levels in human samples for assessment of vitamin D sufficiency. It does not provide therapy or treatment to a patient.

Diagnostic

Yes

The device quantitatively determines 25-hydroxyvitamin D in human serum or EDTA-plasma to assess vitamin D sufficiency, and its results are used in conjunction with other clinical data to assist clinicians in making patient management decisions. These functions are characteristic of a diagnostic device.

SaMD (Software as a Medical Device)

No

The device description clearly outlines a complete kit including reagents, solid phase extraction columns, and requires user-supplied standard laboratory equipment (centrifuge, test tubes, pipettes, etc.) and specific hardware components like an HPLC system with an ESA EC detector, guard and analytical columns, and a dual coulometric EC cell. This is a hardware-based in vitro diagnostic device, not a software-only medical device.

IVD (In Vitro Diagnostic)

Yes, this device is an IVD (In Vitro Diagnostic).

Here's why:

Intended Use: The intended use explicitly states it's for the "quantitative determination of 25-hydroxyvitamin D in human serum or EDTA-plasma". This is a measurement performed in vitro (outside the body) on a biological sample.
Sample Type: It uses "human serum or EDTA-plasma", which are biological specimens.
Purpose: The purpose is to "be used in the assessment of vitamin D sufficiency" and "assist the clinician in making individual patient management decisions". This indicates a medical purpose for the test results.
Device Description: The description details a laboratory test method involving sample preparation, extraction, and analysis using HPLC with electrochemical detection. This is a typical setup for an in vitro diagnostic assay.

The definition of an IVD is a medical device that is used to perform tests on samples such as blood, urine, or tissues to detect diseases or other conditions. This device clearly fits that definition.

PCCP (Predetermined Change Control Plan) Authorized

N/A

Overview

Intended Use / Indications for Use

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.
Indication For Use: K072536

Product codes (comma separated list FDA assigned to the subject device)

MRG

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 200uL 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.

Mentions image processing

Not Found

Mentions AI, DNN, or ML

Not Found

Input Imaging Modality

Not Found

Anatomical Site

Not Found

Indicated Patient Age Range

Adult population. This assay should therefore not be used for measurement in samples from patients

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.

Summary

0

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 Road
Chelmsford, MA 01824
USA
Phone: 978-250-7000
Fax: 978-250-7090 |
|---------------------------------|-------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------|
| Contact Person: | Harold Asp
Quality Assurance Manager |
| Date of Summary
Preparation: | 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 Assay
510(k) 032844 |
| Statement of
Intended Use: | 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. |

1

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.

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

2

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.

3

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.

4

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

5

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

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.

7

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 Test
Concentration | 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 Test
Concentration | Comments |
|---------------|----------------------------|----------------------------------------------------------|
| Paricalcitrol | 100 ng/mL | Approx 100 times therapeutic
range 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.,