The BioPlex 2200 25-OH Vitamin D Kit is a multiplex flow competitive immunoassay intended for the quantitative determination of 25-hydroxyvitamin D in human serum. The BioPlex 2200 25-OH Vitamin D assay is to be used as an aid in the assessment of vitamin D sufficiency.

The BioPlex 2200 25-OH Vitamin D kit is intended for use with the Bio-Rad BioPlex 2200 System.

Device Description

BioPlex 2200 25-OH Vitamin D kit includes the following components:

One (1) 10 mL vial of Bead Set containing dyed beads coated with anti-25-0H ● Vitamin D antibody (sheep), an Internal Standard bead (ISB), and a Serum Verification bead (SVB) in buffer with protein stabilizers (bovine). ProClin 950 (< 1.0%) and sodium azide (< 0.1%) as preservatives.
One (1) 10 mL vial of Release Buffer containing 25-OH Vitamin D releasing reagents in citrate and tri sodium citrate acid buffer at pH 4.1 and ProClin 950 (<1.0%) as preservative.
One (1) 5 mL vial of Conjugate 1 containing biotinylated 25-OH Vitamin D . conjugate and biotinylated anti-human FXIII antibody conjugate (murine) in buffer with protein stabilizers (bovine). ProClin 950 (< 1.0%) and 5-bromo-5nitro-1, 3-dioxane (<0.1%) as preservatives and chemical blockers.
. One (1) 5 mL vial of Conjugate 2 containing phycoerythrin conjugated streptavidin (SA-PE) in a buffer comprising protein stabilizers (bovine). ProClin 950 (< 1.0%) and sodium azide (< 0.1%) as preservatives, chemical blockers and detergent (Tween 20).

Additional materials required but not supplied include BioPlex 2200 Sheath Fluid containing Phosphate Buffered Saline (PBS), ProClin 300 (0.03%) and sodium azide (<0.1%) as preservatives; and BioPlex 2200 Wash Solution containing Phosphate Buffered Saline (PBS) and Tween 20. ProClin 300 (0.03%) and sodium azide (<0.1%) as preservatives.

AI/ML Overview

The BioPlex 2200 25-OH Vitamin D Kit is a multiplex flow competitive immunoassay intended for the quantitative determination of 25-hydroxyvitamin D in human serum, to be used as an aid in the assessment of vitamin D sufficiency. The device underwent a 510(k) submission, K180577, to demonstrate substantial equivalence to its predicate device, BioPlex 2200 25-OH Vitamin D Kit (K141114). The new kit has been modified by re-assigning calibrators and standardizing the assay in accordance with the Vitamin D Standardization Program (VDSP).

Here's an analysis of the acceptance criteria and the studies performed:

1. Table of Acceptance Criteria and Reported Device Performance:

The document primarily focuses on demonstrating substantial equivalence to a predicate device and adherence to CLSI guidelines for analytical performance. Therefore, "acceptance criteria" for specific performance metrics are often implied by the standards set forth in these guidelines and the performance of the predicate device, rather than explicit numerical targets stated as acceptance criteria in the summary. However, we can infer some criteria from the presented data.

Acceptance Criteria (Inferred/Implied)	Reported Device Performance (BioPlex 2200 25-OH Vitamin D Kit)
Precision
Within-run, Between-run, Between-day, Total precision within acceptable limits (CLSI EP5-A3)	Demonstrated with %CVs ranging from 3.0% to 10.7% depending on concentration and type of precision. Most values below 10%.
Reproducibility
Reproducibility across runs/days within acceptable limits (CLSI EP15-A3)	Demonstrated with %CVs ranging from 3.5% to 10.0% depending on concentration and type of precision.
Linearity/Reportable Range
Linearity across the measuring range (CLSI EP06-A)	Demonstrated with good linear regression (e.g., slope 1.0002, r² 0.9956 for one example). Reportable range: 7.0 – 160.0 ng/mL.
Traceability
Traceable to ID-LC/MS/MS RMP and NIST SRM 2972a	Directly stated: "traceable to the 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) 2972a."
Kit Stability
Unopened: 24 months at 2 to 8°C	24 months or until expiration date.
Open: 60 days	60 days.
Sample Stability
Fresh (2 to 8°C): 7 days	7 days.
Frozen (-20 or -70°C): 24 months	24 months.
Freeze-thaw cycles: acceptable	Up to 5 cycles at -20°C, 2 cycles at -70°C.
Detection Limits (LoB, LoD, LoQ)
LoB, LoD, LoQ established (CLSI EP17-A2)	LoB: 5.4 ng/mL, LoD: 7.0 ng/mL, LoQ: 7.0 ng/mL.
Analytical Specificity (Interference)
No significant interference from tested substances at specified concentrations (CLSI EP07-A2)	"No interference was observed with any of the substances tested" at the maximum levels listed (e.g., Hemoglobin <150 mg/dL, Bilirubin <20 mg/dL).
Analytical Specificity (Cross-Reactivity)
Cross-reactivity within acceptable limits	Varied depending on substance (e.g., 25-hydroxyvitamin D2: 93.2%, 1,25-dihydroxyvitamin D3: >100%). Normalized 25(OH) D2 cross-reactivity is 103% (to 25(OH)D3).
Method Comparison with Predicate Device
Good correlation and agreement with predicate device (CLSI EP09-A3)	Slope: 1.048 (95% CI: 1.009 - 1.088), Intercept: -0.885 (95% CI: -2.211 - 0.441), Correlation Coefficient (r): 0.987 (95% CI: 0.983 - 0.990).
Method Comparison to Reference Method (VDSP-certified RMP)
Good correlation and agreement with VDSP RMP	Slope: 0.993 (95% CI: 0.913 - 1.073), Intercept: -0.775 (95% CI: -2.705 - 1.155), Correlation Coefficient (r): 0.949 (95% CI: 0.927 - 0.964).
Bias at Medical Decision Levels
Acceptable bias at key medical decision points (20, 30, 100 ng/mL)	Mean bias: -7.3% at 20 ng/mL, -0.2% at 30 ng/mL, 3.5% at 100 ng/mL. Overall mean bias: 1.0%.
Expected Values/Reference Range
Established from a healthy population (CLSI EP28-A3c)	Mean: 31.9 ng/mL, Median: 29.2 ng/mL, 2.5th – 97.5th percentile: 14.0 – 76.3 ng/mL (based on 288 healthy donors).

2. Sample size used for the test set and data provenance:

Precision Studies (CLSI EP5-A3):
- Sample Size: 6 human serum samples and 2 control levels. Each tested in duplicate per run, on two runs per day over 20 days (N=80 results per sample/control).
- Data Provenance: Not explicitly stated (e.g., country of origin, retrospective/prospective), but it was human serum from a "panel".
Reproducibility Studies (CLSI EP15-A3):
- Sample Size: 8 panel members (serum matrix and QC controls), tested in replicates of two on two runs per day over five days (20 replicates per panel member).
- Data Provenance: Performed at "one (1) US testing facility." Not explicitly stated if retrospective or prospective, or specific origin of serum matrix.
Linearity/Assay Reportable Range (CLSI EP06-A):
- Sample Size: Five high patient serum samples initially, serially diluted. Each sample and dilution evaluated in replicates of four.
- Data Provenance: Not explicitly stated.
Detection Limits (LoB, LoD, LoQ) (CLSI EP17-A2):
- LoB: Five blank samples, tested in 4 replicates per day for 5 days (100 data points per reagent lot).
- LoD: Six human samples with low 25-OH vitamin D, tested in 10 replicates per day for five days (50 data points per sample per reagent lot).
- Data Provenance: Not explicitly stated.
Interfering Substances (CLSI EP07-A2):
- Sample Size: Not explicitly stated for number of samples, but "human serum pools" were used.
- Data Provenance: Not explicitly stated.
Cross-Reactivity (CLSI EP17-A2):
- Sample Size: 2 human serum pools. Nine cross-reactants were spiked into these pools. Spiked and non-spiked samples evaluated in replicates of five.
- Data Provenance: Not explicitly stated.
Method Comparison with Predicate Device (CLSI EP09-A3):
- Sample Size: 201 human samples (182 unaltered, 19 spiked with 25-hydroxyvitamin D3).
- Data Provenance: Not explicitly stated (e.g., country of origin, retrospective/prospective).
Method Comparison to Reference Method (VDSP):
- Sample Size: 120 native single donor patient serum samples.
- Data Provenance: "reference sample set provided by the Vitamin D Standardization and Certification Program with assigned values by the RMP at CDC, independent from the samples used for standardization." This indicates a high-quality, external reference set. The origin of the patient samples themselves is not specified beyond "native single donor patient serum samples".
Bias at Vitamin D Medical Decision Levels:
- Sample Size: 303 samples overall for bias analysis, with specific numbers at each decision range (e.g., 7 for 19-21 ng/mL, 20 for 29-31 ng/mL, 6 for 95-105 ng/mL).
- Data Provenance: Presumably a subset or re-analysis of samples from the method comparison studies.
Expected Values/Reference Range (CLSI EP28-A3c):
- Sample Size: 288 samples from "apparently healthy donors" (161 males, 127 females).
- Data Provenance: Samples collected from "three regions (Northern, Central, and Southern) in the US" in spring, summer, and winter. Included African Americans, Hispanics, and Caucasians. This is prospective collection for establishing a reference range.

3. Number of experts used to establish the ground truth for the test set and the qualifications of those experts:

This device is an in vitro diagnostic (IVD) quantitative assay for a biomarker (25-OH Vitamin D). The "ground truth" for such devices is established through highly accurate and standardized analytical methods.

For the Method Comparison to Reference Method, the ground truth was established by the Vitamin D Standardization and Certification Program (VDSP) using an ID-LC/MS/MS Reference Method Procedure (RMP) at CDC. This RMP is traceable to the National Institute of Standards and Technology (NIST) Standard Reference Material (SRM) 2972a. These are highly qualified and internationally recognized reference methods and institutions for chemical measurements, representing the gold standard in analytical chemistry. It is not about expert consensus on interpretations, but rather expert execution and certification of analytical accuracy.
For other studies (precision, linearity, etc.), the "ground truth" is typically the measured value itself by the reference method or the assigned value of controls and calibrators, which are also traceable to established analytical standards.

4. Adjudication method for the test set:

Not applicable in the conventional sense involving multiple human readers and an adjudication process. This is a quantitative IVD device where sample values are measured against established reference methods and standards, not interpreted by experts in a clinical imaging or diagnostic context that would require adjudication.

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:

Not applicable. This is a laboratory diagnostic assay, not an AI-assisted diagnostic tool that involves human readers interpreting cases.

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

This device performs as a standalone automated multiplex flow competitive immunoassay on the Bio-Rad BioPlex 2200 System. Its performance characteristics (precision, linearity, LLoQ, etc.) were evaluated directly without human intervention in the measurement process, representing a "standalone" analytical performance assessment. However, it's not an "algorithm only" device in the sense of AI; it's a chemical assay. The output is a quantitative measure of 25-OH Vitamin D concentration.

7. The type of ground truth used:

For method comparison, the ground truth for the 120 samples was assigned values by the RMP at CDC, provided by the Vitamin D Standardization and Certification Program (VDSP). This is a highly accurate and externally certified analytical ground truth.
For other analytical validation studies, the ground truth is established through internal standards, traceable calibrators, and reference materials specified by CLSI guidelines and traceable to NIST SRM 2972a.

8. The sample size for the training set:

This submission describes performance validation for a modified in vitro diagnostic kit. The "training set" concept (as used in machine learning) is not directly applicable here. The device itself (the BioPlex 2200 system and its reagents) has established methodologies.
The document states that Bio-Rad "modified the kit by re-assigning the calibrators and standardizing the assay in accordance with the Vitamin D Standardization Program (VDSP)." This implies that Bio-Rad used samples and measurements to establish these new calibrator assignments and standardization. However, the specific size and nature of such internal development/calibration "training" sets are not detailed in this 510(k) summary. The summary focuses on the validation of the finalized device.

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

Again, the concept of a "training set" for an IVD kit often refers to the samples used during development and calibration. For this device, the key aspect of this "ground truth" for the modified kit is its standardization in accordance with the Vitamin D Standardization Program (VDSP). This means that the calibrators and ultimately the assay's measurements are referenced to the same highly accurate ID-LC/MS/MS RMP and NIST SRM 2972a that were used to establish the ground truth for the external validation sample sets. This ensures analytical accuracy and comparability across different methods and laboratories. The actual process of selecting and assigning values to the new calibrators would involve measuring reference materials and/or highly characterized samples using the VDSP-traceable methods.

Summary

{0}------------------------------------------------

Image /page/0/Picture/2 description: The image shows the logo of the U.S. Food and Drug Administration (FDA). On the left is the seal of the Department of Health & Human Services. To the right of the seal is the FDA logo, which consists of the letters "FDA" in a blue square, followed by the words "U.S. FOOD & DRUG" in blue, and the word "ADMINISTRATION" in a smaller font below.

June 22, 2018

Bio-Rad Laboratories Arlene Carillo Regulatory Affairs Specialist 5500 East Second Street Benicia. CA 94510

Re: K180577

Trade/Device Name: BioPlex 2200 25-OH Vitamin D Kit Regulation Number: 21 CFR 862.1825 Regulation Name: Vitamin D test system Regulatory Class: Class II Product Code: MRG Dated: May 15, 2018 Received: May 16, 2018

Dear Arlene Carillo:

We have reviewed your Section 510(k) premarket notification of intent to market the device referenced above and have determined the device is substantially equivalent (for the indications for use stated in the enclosure) to legally marketed predicate devices marketed in interstate commerce prior to May 28, 1976, the enactment date of the Medical Device Amendments, or to devices that have been reclassified in accordance with the provisions of the Federal Food, Drug, and Cosmetic Act (Act) that do not require approval of a premarket approval application (PMA). You may, therefore, market the device, subject to the general controls provisions of the Act. The general controls provisions of the Act include requirements for annual registration, listing of devices, good manufacturing practice, labeling, and prohibitions against misbranding and adulteration. Please note: CDRH does not evaluate information related to contract liability warranties. We remind you, however, that device labeling must be truthful and not misleading.

If your device is classified (see above) into either class II (Special Controls) or class III (PMA), it may be subject to additional controls. Existing major regulations affecting your device can be found in the Code of Federal Regulations, Title 21, Parts 800 to 898. In addition, FDA may publish further announcements concerning your device in the Federal Register.

Please be advised that FDA's issuance of a substantial equivalence determination does not mean that FDA has made a determination that your device complies with other requirements of the Act or any Federal statutes and regulations administered by other Federal agencies. You must comply with all the Act's requirements, including, but not limited to: registration and listing (21 CFR Part 807); labeling (21 CFR Part 801 and Part 809); medical device reporting of medical device-related adverse events) (21 CFR 803); good manufacturing practice requirements as set forth in the quality systems (QS) regulation (21 CFR

{1}------------------------------------------------

Part 820); and if applicable, the electronic product radiation control provisions (Sections 531-542 of the Act); 21 CFR 1000-1050.

Also, please note the regulation entitled, "Misbranding by reference to premarket notification" (21 CFR Part 807.97). For questions regarding the reporting of adverse events under the MDR regulation (21 CFR Part 803), please go to http://www.fda.gov/MedicalDevices/Safety/ReportaProblem/default.htm.

For comprehensive regulatory information about mediation-emitting products, including information about labeling regulations, please see Device Advice (https://www.fda.gov/MedicalDevices/DeviceRegulationandGuidance/) and CDRH Learn (http://www.fda.gov/Training/CDRHLearn). Additionally, you may contact the Division of Industry and Consumer Education (DICE) to ask a question about a specific regulatory topic. See the DICE website (http://www.fda.gov/DICE) for more information or contact DICE by email (DICE@fda.hhs.gov) or phone (1-800-638-2041 or 301-796-7100).

Sincerely,

Kellie B. Kelm -S

for Courtney H. Lias, Ph.D. Director Division of Chemistry and Toxicology Devices Office of In Vitro Diagnostics and Radiological Health Center for Devices and Radiological Health

Enclosure

{2}------------------------------------------------

Indications for Use

510(k) Number (if known) K180577

Device Name BioPlex 2200 25-OH Vitamin D Kit

Indications for Use (Describe)

BioPlex 2200 25-OH Vitamin D Kit

The BioPlex 2200 25-OH Vitamin D kit is intended for use with the Bio-Rad BioPlex 2200 System.

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

☑ Prescription Use (Part 21 CFR 801 Subpart D)
☐ Over-The-Counter Use (21 CFR 801 Subpart C)

CONTINUE ON A SEPARATE PAGE IF NEEDED.

This section applies only to requirements of the Paperwork Reduction Act of 1995.

DO NOT SEND YOUR COMPLETED FORM TO THE PRA STAFF EMAIL ADDRESS BELOW.

The burden time for this collection of information is estimated to average 79 hours per response, including the time to review instructions, search existing data sources, gather and maintain the data needed and complete and review the collection of information. Send comments regarding this burden estimate or any other aspect of this information collection, including suggestions for reducing this burden, to:

Department of Health and Human Services Food and Drug Administration Office of Chief Information Officer Paperwork Reduction Act (PRA) Staff PRAStaff@fda.hhs.gov

"An agency may not conduct or sponsor, and a person is not required to respond to, a collection of information unless it displays a currently valid OMB number."

{3}------------------------------------------------

BioPlex 2200 25-OH Vitamin D 510(k) Summary

Bio-Rad Laboratories hereby submits this 510(k) in accordance with the requirements of SMDA 1990 and 21 CFR 807.92. This summary of 510(k) safety and effectiveness information provides detail as a basis for a determination of substantial equivalence for the BioPlex 2200 25-OH Vitamin D kit. The BioPlex 2200 25-OH Vitamin D was originally cleared under K141114. Bio-Rad has modified the kit by re-assigning the calibrators and standardizing the assay in accordance with the Vitamin D Standardization Program (VDSP).

510(k) Number: K180577

Summary Preparation Date: June 18, 2018

Applicant: Bio-Rad Laboratories

Contact:

Arlene Carillo Regulatory Affairs Specialist 5500 East Second Street Benicia, CA 94510

Measurand:

25-hydroxyvitamin D

Type of Test:

Quantitative multiplexed flow immunoassay

Proprietary and Established Names:

BioPlex 2200 25-OH Vitamin D

Regulatory Information:

1. Regulation section: 21 CFR §862.1825 – Vitamin D test system
1. Product code: MRG, System, Test, Vitamin D

{4}------------------------------------------------

3. Panel:

Clinical Chemistry (75)

Intended Use:

1. Intended use(s):
  The BioPlex 2200 25-OH Vitamin D Kit is a multiplex flow competitive immunoassay intended for the quantitative determination of 25-hydroxyvitamin D in human serum. The BioPlex 2200 25-OH Vitamin D assay is to be used as an aid in the assessment of vitamin D sufficiency.

The BioPlex 2200 25-OH Vitamin D kit is intended for use with the Bio-Rad BioPlex 2200 System.

1. Indication(s) for use: Same as Intended Use
1. Special conditions for use statement(s): For prescription use only
Special instrument requirements: 4. Bio-Rad BioPlex 2200 System

Device Description:

BioPlex 2200 25-OH Vitamin D kit includes the following components:

One (1) 10 mL vial of Bead Set containing dyed beads coated with anti-25-0H ● Vitamin D antibody (sheep), an Internal Standard bead (ISB), and a Serum Verification bead (SVB) in buffer with protein stabilizers (bovine). ProClin 950 (< 1.0%) and sodium azide (< 0.1%) as preservatives.
One (1) 10 mL vial of Release Buffer containing 25-OH Vitamin D releasing reagents in citrate and tri sodium citrate acid buffer at pH 4.1 and ProClin 950 (<1.0%) as preservative.
One (1) 5 mL vial of Conjugate 1 containing biotinylated 25-OH Vitamin D . conjugate and biotinylated anti-human FXIII antibody conjugate (murine) in buffer with protein stabilizers (bovine). ProClin 950 (< 1.0%) and 5-bromo-5nitro-1, 3-dioxane (<0.1%) as preservatives and chemical blockers.
. One (1) 5 mL vial of Conjugate 2 containing phycoerythrin conjugated streptavidin (SA-PE) in a buffer comprising protein stabilizers (bovine). ProClin 950 (< 1.0%) and sodium azide (< 0.1%) as preservatives, chemical blockers and detergent (Tween 20).

Additional materials required but not supplied include BioPlex 2200 Sheath Fluid

{5}------------------------------------------------

containing Phosphate Buffered Saline (PBS), ProClin 300 (0.03%) and sodium azide (<0.1%) as preservatives; and BioPlex 2200 Wash Solution containing Phosphate Buffered Saline (PBS) and Tween 20. ProClin 300 (0.03%) and sodium azide (<0.1%) as preservatives.

Substantial Equivalence Information:

1. Predicate device name(s): BioPlex 2200 25-OH Vitamin D Kit, K141114
1. Comparison with predicate:

Characteristics	New DeviceBioPlex 2200 25-OHVitamin D Kit	Predicate DeviceBioPlex 2200 25-OHVitamin D Kit,K141114
Intended Use	multiplex flowcompetitiveimmunoassay intendedfor the quantitativedetermination of 25-hydroxyvitamin D.	Same
Indications for Use	To be used as an aid inthe assessment ofvitamin D sufficiency.	Same
Measured Analyte	25-hydroxyvitamin D	Same
Assay Type	Quantitative	Same
Assay Technology	Automated multiplexflow competitiveimmunoassay	Same
Test Principle	Competitiveimmunoassay	Same
Antibody	Sheep antibody against25 OH Vitamin D	Same
Solid Phase	Antibody-coatedparamagneticmicrobeads	Same
Signal Detection	Fluorescence	Same

{6}------------------------------------------------

Device Similarities
Characteristics	New DeviceBioPlex 2200 25-OHVitamin D Kit	Predicate DeviceBioPlex 2200 25-OHVitamin D Kit,K141114
Conjugate	Biotinylated 25-hydroxyvitamin D andphycoerythrinconjugated streptavidin	Same
Reagent IntegralStorage	On-board or inrefrigerator at 2-8°C	Same
SampleHandling/Process	Automated	Same
Unit of Measure	ng/mL	Same
Sample Matrix	Serum	Same
Sample Size	10 μL	Same
Calibrator(s) andCalibration	6 calibrator levels (soldseparately); 4-PL(parameter logistic)curve fit algorithm	Same
Open Pack Stability	60 days	Same
Reagent PackCalibrationFrequency	Every 30 days	Same
Instrumentation	Bio-Rad BioPlex 2200System	Same

Device Differences
Characteristics	New DeviceBioPlex 2200 25-OHVitamin D Kit	Predicate DeviceBioPlex 2200 25-OHVitamin D Kit,K141114
Measuring range	7.0 – 160.0 ng/mL	6.5 – 125.0 ng/mL
Calibrator Levels	Level 1: 0.0 ng/mLLevel 2: 10.0 ng/mLLevel 3: 30.0 ng/mLLevel 4: 70.0 ng/mLLevel 5: 110.0 ng/mLLevel 6: 180.0 ng/mL	Level 1: 0.0 ng/mLLevel 2: 10.0 ng/mLLevel 3: 30.0 ng/mLLevel 4: 75.0 ng/mLLevel 5: 110.0 ng/mLLevel 6: 165.0 ng/mL
Traceable to:	Internalstandards which are traceableto CDC and GhentUniversity's ID-LC-MS/MS 25 OH vitamin DReference Method Procedure	Internal standard(stock) using UVabsorbancespectrometry

{7}------------------------------------------------

Device Differences
Characteristics	New Device	Predicate Device
	BioPlex 2200 25-OHVitamin D Kit	BioPlex 2200 25-OHVitamin D Kit,K141114
	(RMP) that is traceable to theNational Institute ofStandards and TechnologyNIST SRM 2972a

Standard/Guidance Document Referenced (if applicable):

EP05-A3, Evaluation of Precision Performance of Quantitative Measurement Procedures; Approved Guideline, Third Edition (Vol. 34 No.13)

EP06-A, Evaluation of Linearity of Quantitative Measurement: A Statistical Approach, Approved Guideline (Vol. 23 No.16)

EP07-A2. Interference Testing in Clinical Chemistry, Approved Guideline, Second Edition (Vol. 25 No.27)

EP09-A3. Measurement Procedure Comparison and Bias Estimation Using Patient Samples: Approved Guideline - Third Edition (Vol. 33 No. 11)

EP15-A3, User Verification of Precision and Estimation of Bias, Approved Guideline, Third Edition (Vol. 34 No.12)

EP17-A2, Evaluation of Detection Capability for Clinical Laboratory Measurement Procedures; Approved Guideline, Second Edition (Vol. 32 No.8)

EP28-A3c, Defining, Establishing, and Verifying Reference Intervals in the Clinical Laboratory; Approved Guideline, Third Edition (Vol. 28 No.30)

Test Principle:

The BioPlex 2200 25-OH Vitamin D assay is a multiplex flow competitive immunoassay for the quantitative determination of 25-hydroxyvitamin D in human serum.

A population of dyed paramagnetic beads is coated with anti-25-OH Vitamin D antibody. The BioPlex 2200 System combines an aliquot of patient sample with the Vitamin D Release Buffer to dissociate the 25-OH Vitamin D from its binding protein. After the first incubation, the bead reagent is added to the reaction vessel and incubated at 37°C. After the second incubation, the BioPlex 2200 system adds the 25-OH Vitamin D-Biotin conjugate 1. The excess conjugate 1 is removed during a wash cycle and the streptavidin-phycoerythrin (SA-PE) conjugate 2 is added. The excess conjugate 2 is removed during a wash cycle, and the beads are re-suspended in sheath fluid. The bead mixture then passes through the detector. The detected fluorescence of the SA-PE in relative fluorescence intensity (RFI) is inversely proportional to the concentration of 25-OH Vitamin D in the sample. Two additional dyed beads, an Internal Standard Bead (ISB) and a Serum Verification Bead (SVB), are present in each reaction mixture to verify detector response and the addition of serum to the reaction vessel.

{8}------------------------------------------------

The BioPlex 2200 25-OH Vitamin D assay is standardized (calibrated) using internal standards which are traceable to the ID-LC/MS/MS 25(OH) vitamin D Reference Method Procedure (RMP). The ID-LC/MS/MS is traceable to the National Institute of Standards and Technology (NIST) Standard Reference Material (SRM) 2972a.

The BioPlex 2200 25-OH Vitamin D results are each expressed in ng/mL or nmol/L.

Performance Characteristics (if/when applicable):

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

Precision Studies

Precision testing of the BioPlex 2200 25-OH Vitamin D kit was performed on the BioPlex 2200 instrument in accordance with CLSI EP5-A3 guideline. A human serum panel consisting of 6 samples spanning the measuring range and two levels of the BioPlex 2200 25-OH Vitamin D controls were assayed in duplicate per run on two runs per day over 20 days (N=80) with one reagent lot. The data were analyzed for within-run, between-run, between-day, and total precision and the mean (ng/mL), standard deviation (ng/mL) and percent coefficient of variation (%CV) are summarized below. SD was evaluated for samples with concentrations ≤ 25 ng/mL and %CV was used for samples with concentrations > 25 ng/mL.

SerumPanel	N	Mean(ng/mL)	Within Run		Between-Run		Between-Day		Total Precision
			SD	%CV	SD	%CV	SD	%CV	SD	%CV
Sample 1	80	16.4	1.02	6.2	1.19	7.2	0.45	2.7	1.62	9.9
Sample 2	80	18.9	1.36	7.2	0.76	4.0	0.73	3.9	1.72	9.1
Sample 3	80	37.2	1.66	4.4	1.61	4.3	0.00	0.0	2.31	6.2
Sample 4	80	46.5	3.66	7.9	3.07	6.6	1.35	2.9	4.96	10.7
Sample 5	80	81.4	2.79	3.4	3.42	4.2	0.00	0.0	4.41	5.4
Sample 6	80	100.4	3.89	3.9	5.34	5.3	0.00	0.0	6.60	6.6
Control 1	80	21.5	0.88	4.1	1.75	8.1	0.00	0.0	1.96	9.1
Control 2	80	54.1	1.62	3.0	2.45	4.5	0.00	0.0	2.94	5.4

BioPlex 2200 25-OH Vitamin D - CLSI EP5-A3 Precision

Reproducibility Studies

A reproducibility panel, consisting of eight (8) panel members made using serum matrix and BioPlex 2200 25-OH Vitamin D QC controls were tested in replicates of two (2) on two (2) runs per day over five (5) days at one (1) site (2 replicates x 2 run x 5 days x 1 Site = 20 replicates per panel member). The serum sample panel includes two (2) samples representing the following concentrations: 7 and 20 ng/mL, 21 and 40 ng/mL, 41 and 80 ng/mL and 81 and 150 ng/mL.

{9}------------------------------------------------

Reproducibility testing was performed at one (1) US testing facility using one (1) lot of the BioPlex 2200 25-OH Vitamin D Reagent Pack, one (1) lot of BioPlex 2200 25-OH Vitamin D Calibrator Set and one (1) lot of BioPlex 2200 25-OH Vitamin D Control Set. The data were analyzed for within-run (repeatability), between-run, between-day, between-site and total precision according to the principles described in Clinical Laboratory Standards Institute (CLSI) EP15-A3.

Serum Panel	N	Mean (ng/mL)	Within-Run		Between-Run		Between-Day		Total
			SD	%CV	SD	%CV	SD	%CV	SD	%CV
Sample 1	20	15.9	0.96	6.0	0.72	4.5	0.00	0.0	1.20	7.6
Sample 2	20	17.3	1.22	7.0	0.90	5.2	0.00	0.0	1.52	8.8
Sample 3	20	23.7	2.22	9.4	0.00	0.0	0.84	3.6	2.37	10.0
Sample 4	20	25.5	1.49	5.8	1.29	5.1	0.00	0.0	1.97	7.7
Sample 5	20	52.4	1.86	3.5	1.82	3.5	1.74	3.3	3.13	6.0
Sample 6	20	56.5	3.58	3.7	0.00	0.0	1.88	3.3	4.04	7.1
Sample 7	20	88.9	3.66	4.1	0.00	0.0	1.64	1.8	4.01	4.5
Sample 8	20	94.9	4.27	4.5	0.00	0.0	0.57	0.6	4.31	4.5
QC Control L1	20	23.4	1.28	5.5	1.06	4.5	0.00	0.0	1.67	7.1
QC Control L2	20	67.9	2.04	3.0	0.00	0.0	2.46	3.6	3.19	4.7
Range(SD:≤25ng/mL;%CV:>25ng/mL)		15.9 - 94.9	0.96 -2.22	3.5 -5.8	0.00 -1.06	0.0 -5.1	0.00 -0.84	0.0 -3.6	1.20 -2.37	4.5 -7.7

BioPlex 2200 25-OH Vitamin D - CLSI EP15-A3 Reproducibility

b. Linearity/assay reportable range:

Five high patient serum samples with 20% higher than upper limit of the assay range were tested to demonstrate linearity. These samples were serially diluted with low levels of human sample near LoQ in accordance with CLSI EP06-A guideline. Each sample and dilution was evaluated in replicates of four using one BioPlex 2200 25-OH Vitamin D reagent lot on one instrument. Linear and polynomial regression analysis of 25-OH Vitamin D recovery vs. sample dilution was performed to determine if the dilution curves exhibit statistically significant non-linear regression based on the CLSI guideline EP06-A.

See one example below for the regression parameters (slope, intercept and r') of the observed values vs. predicted values.

Concentration(ng/mL)	Slope	Intercept	r2	Dilution Range(ng/mL)
166.5	1.0002	0.0143	0.9956	5.4-166.5

The BioPlex 2200 25-OH Vitamin D assay has demonstrated that the assay range supports up to 160.0 ng/mL.

Over-Range (OR) results may be generated for values greater than the reportable measuring range and results are reported as > 160.0 ng/mL.

{10}------------------------------------------------

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

Calibrator Traceability

The BioPlex 2200 25-OH Vitamin D Calibrators are traceable to the liquid chromatography tandem mass spectrometry (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) 2972a.

Kit Stability:

BioPlex 2200 25-OH Vitamin D Kit: Real Time (unopened) Kit Stability, 24 months or until the date of expiration when stored unopened on the instrument or at 2 to 8℃; the open kit claim is 60 days.

Sample Stability:

Fresh sample stability (2 to 8°C), 7 days; Frozen sample stability (-20 or -70°C), 24 months; Sample Freeze-thaw (-20 or -70°C), up to 5 freeze thaw cycles at -20℃ and 2 freeze thaw cycles at -70℃ acceptable.

d. Detection limit:

The study was conducted in accordance with CLSI EP17-A2 guideline for determining the Limit of Blank (LoB), Limit of Detection (LoD) and Limit of Quantitation (LoQ).

Limit of Blank (LoB)

Five blank samples were tested with two BioPlex 2200 25-OH Vitamin D reagent lots on one instrument in 4 replicates per day for 5 days yielding 100 data points per reagent lot.

A non-parametric statistical analysis at 95th percentile is used to calculate LoB.

Limit of Detection (LoD)

Six human samples with low level of 25-OH vitamin D in the range of 5 to 30 ng/mL were tested with two BioPlex 25-OH Vitamin D reagent lots on one instrument in 10 replicates per day for five days yielding 50 data points per sample per reagent lot. LoD is then calculated by the equation:

LoD = LoB + c , SD + c , SD is a multiplier to give the 95th percentile of a normal distribution and SD is from the linear regression of standard deviation versus 25-OH Vitamin D mean value.

Limit of Quantitation (LoQ)

The LoQ was evaluated based on the accuracy goal which was defined as precision ≤ 20% CV. The %CV was calculated using the same measurement results of the 6 low level samples used for determining the LoD.

{11}------------------------------------------------

LoB	LoD	LoQ
5.4	7.0	7.0

The results of LoB, LoD, and LoQ in ng/mL are summarized in the table below.

e. Analytical specificity:

Interfering Substances

An interfering substances study was conducted to evaluate the potential interference of specific endogenous and exogenous substances with the BioPlex 2200 25-OH Vitamin D kit according to CLSI EP07-A2 guideline. No interference was observed with any of the substances tested.

The substances and the maximum levels tested are shown in the table below:

Substance	Concentration
Hemoglobin	<150 mg/dL
Bilirubin (unconjugated)	<20 mg/dL
Bilirubin (conjugated)	<30 mg/dL
Triglycerides	<350 mg/dL
Total Protein	<12 g/dL
Cholesterol	<450 mg/dL
Uric Acid	<20 mg/dL
HAMA	≤100 ng/mL
Rheumatoid Factor	<350 IU/mL
Ascorbic Acid	<3 mg/dL
EDTA	<800 mg/dL
Heparin Lithium	≤ 8000 units/dL
Heparin Sodium	≤ 8000 units/dL
Biotin	<3600 ng/mL

Cross-Reactivity

The study was conducted in accordance with CLSI EP17-A2 using 2 human serum pools at 25-hydroxyvitamin D concentrations of 31.5 and 43.2 ng/mL. Nine cross reactants at levels listed below were then spiked into the human serum pools. The spiked and non-spiked samples were evaluated in replicates of five to calculate the cross reactivity as shown below.

% Cross Reactivity = (spiked vitamin D - non-spiked vitamin D) = Cross reactant concentration × 100%

The results of each potential cross reactant are listed below.

Cross Reactant	SpikedConcentration	% CrossReactivity
----------------	-------------------------	-----------------------

{12}------------------------------------------------

	(ng/mL)
25-hydroxyvitamin D2	30	93.2%
25-hydroxyvitamin D3	30	90.8%
Vitamin D2	1000	0.3%
Vitamin D3	1000	0.3%
1,25-dihydroxyvitamin D2	30	92.2%
1,25-dihydroxyvitamin D3	30	>100%
3-epi 25-hydroxyvitamin D3	30	70.8%
24,25-dihydroxyvitamin D3	20	15.5%
Paricalcitol (Zemplar)	24	>100%

To avoid potential under reporting of 25(OH) D2 cross reactivity due to endogenous binding characteristics of the metabolites in a sample, the observed % cross reactivity was normalized to 25(OH) D3. Assuming the normalized 25(OH) D3 value of 100%, the 25(OH) D2 cross reactivity is 103%.

2. Comparison studies:

Method comparison with predicate device: a. Method comparison studies were performed following CLSI EP09-A3 guideline.
A total of two hundred and one (201) human samples spanning the entire measuring assay range were tested in one replicate with both the BioPlex 2200 25-OH Vitamin D kit and the predicate assay. Of the 201 samples, there were 182 unaltered samples and 19 samples spiked with 25-hydroxyvitamin D3 to supplement the assay range. Sample values lower or higher than the measuring range of either the test or comparator method were not included in the analysis. Mean values of 25-OH Vitamin D results were plotted using Weighted Deming regression analysis on the Analyse-it software for all samples spanning the measuring range of both assays. Results of the regression slope, intercept, and coefficient of correlation (r) are summarized in the table below.

Number ofResultsAnalyzed	Slope(95% CI)	Intercept(95% CI)	CorrelationCoefficient (r)(95% CI)	Measured Range(ng/mL)
184	1.048(1.009 - 1.088)	-0.885(-2.211 -0.441)	0.987(0.983 - 0.990)	Candidate: 11.2 -136.2Predicate: 9.1 -122.8

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

b. Method comparison to reference method:

A total of one hundred and twenty (120) native single donor patient serum samples (reference sample set provided by the Vitamin D Standardization and Certification Program with assigned values by the RMP at CDC, independent from the samples used for standardization) spanning the entire measuring assay range were tested with one replicate on the BioPlex 2200 25-OH Vitamin D kit. Sample values lower or higher than the measuring range of the BioPlex 2200 25-OH Vitamin D assay were not included in the analysis. Results of the Weighted Deming regression slope, intercept, and coefficient of correlation (r) are summarized in the table below.

Number ofResultsAnalyzed	Slope(95% CI)	Intercept(95% CI)	CorrelationCoefficient (r)(95% CI)	Measured Range(ng/mL)
119	0.993(0.913 - 1.073)	-0.775(-2.705 - 1.155)	0.949(0.927 - 0.964)	Candidate 9.0 -158.4VDSP: 7.4 - 153.2

Bias at Vitamin D Medical Decision Levels C. Samples at medical decision levels of 20, 30 and 100 ng/mL were analyzed to determine mean bias and 95% confidence interval using Bland-Altman analysis on the Analyse-it software. The data is summarized in the table below.

Medical Decision Range(ng/mL)	Average(ng/mL)	N	Mean Bias	95% CI
19-21	20.0	7	-7.3%	-19.94 – 5.26
29-31	29.7	20	-0.2%	-6.08 - 5.69
95-105	101.7	6	3.5%	-9.76 – 16.80
Overall	41.8	303	1.0%	-0.79 - 2.78

d. Matrix comparison: Serum only
1. Expected values/Reference range:

The Expected Values study was conducted following CLSI EP28-A3c guideline.

Two hundred and eighty-eight (288) samples from apparently healthy donors including 161 males ranging in age from 21 to 79 and 127 females ranging in age from 21 to 66 were collected from three regions (Northern, Central, and Southern) in the US in spring, summer and winter, including African Americans, Hispanics and Caucasians.

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

The 288 samples from apparently healthy donors met the following inclusion/exclusion criteria as follows and tested with the BioPlex 2200 25-OH Vitamin D kit in singlicate.

Age from 21 to 90 ●
. Roughly 50% female and 50% male
20% from Northern, 20% from Central and 60% from Southern region
40% collected in Spring, 30% in Summer and 30% in Winter
. At least 30% African Americans and 30% Caucasians
90% not taking Vitamin D supplements and <30% of those taking Vitamin D supplements at or more than 1000 IU, but less than 2000 IU
. Normal TSH, PTH, and Total Calcium
No family history of parathyroid or calcium regulatory disease. In addition, ● no personal history of kidney disease, GI disease, liver disease, and no bariatric surgery

The observed median, mean, and range between 2.5th to 97.5th percentile are summarized below.

N	Mean	Median	2.5th - 97.5th percentile
288	31.9 ng/mL	29.2 ng/mL	14.0 – 76.3 ng/mL
	79.8 nmol/L*	72.9 nmol/L	35.1 – 190.7 nmol/L

*nmol/L = ng/mL x2.5

Each laboratory should establish its own reference range pertinent to their specific patient populations.

Conclusion:

The submitted information in this 510(k) Premarket Notification will support a determination of substantial equivalence for BioPlex 2200 25-OH Vitamin D. The clinical and non-clinical tests demonstrate that this device is as safe, as effective and performs as well as the legally marketed predicate device.

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.