The LIAISON® XL 1,25 Dihydroxyvitamin D is an in vitro chemiluminescent immunoassay (CLIA) intended for the quantitative determination of 1,25 dihydroxyvitamin (1,25(OH)2D) in serum, EDTA and Lithium Heparin plasma. Results of the 1,25 Dihydroxyvitamin D are used in the assessment of vitamin D sufficiency. Assay results should be used in conjunction with other clinical and laboratory data to assist the clinician in making individual patient management decisions in adult populations. The test is to be performed on the LIAISON® XL Analyzer.

The LIAISON® XL 1,25 Dihydroxyviamin D Control Set is intended for use as assayed quality control samples to monitor the performance of the LIAISON® XL 1,25 Dihydroxyvitamin D assay.

The LIAISON® XL 1.25 Dihydroxyvitamin D Calibration Verifiers are assayed quality control materials intended for the quantitative verification of calibration and reportable range of the LIAISON® XL1,25 Dihydroxyvitamin D assay.

Device Description

The LIAISON® XL 1,25 Dihydroxyvitamin D assay is a modified three-step sandwich assay that uses a recombinant fusion protein for capture of the 1,25 (OH)> D molecule and a murine monoclonal antibody which specifically recognizes the complex formed by the recombinant fusion protein with the 1,25(OH)2 D molecule. Results are determined by a 2 point calibration conversion of the master curve to a working curve. The light signal is measured by a photomultiplier as relative light units (RLU) and is proportional to the concentration of 1,25(OH)> D present in the calibrators, controls or patient samples.

LIAISON® XL 1,25 Dihydroxyvitamin D Control set contains;

2 levels controls containing human serum spiked with 1,25 (OH)2 D, and . preservatives; 2 vials each level; lyophilized
The target concentration for control level 1 is 35 pg/mL. The target concentration for control Level 2 is 120 pg/mL.
The range of concentrations of each control is reported on the certificate of analysis provided with each LIAISON® XL 1.25 Dihydroxyvitamin D Control set.

LIAISON® XL 1,25 Dihydroxyvitamin D Calibration Verifier set contains:

4 levels containing human serum spiked with 1,25 (OH), D, and preservatives, . 1 vial each level, lyophilized
The target concentration for cal verifier A is 15 pg/mL. The target concentration for cal verifier B is 40 pg/mL. The target concentration for cal verifier C is 80 pg/mL. The target concentration for cal verifier D is 150 pg/mL.
The range of concentrations of each calibration verifier is reported on the certificate of analysis provided with each LIAISON® XL 1,25 Dihydroxyvitamin D Calibration Verifier set.

AI/ML Overview

The provided document is a 510(k) Summary for the LIAISON® XL 1,25 Dihydroxyvitamin D assay and its associated control and calibration verifier sets. This summary describes the device's intended use and provides performance characteristics to demonstrate substantial equivalence to a predicate device.

Here's an analysis of the acceptance criteria and study data based on the provided text:

1. A table of acceptance criteria and the reported device performance

The document does not explicitly state "acceptance criteria" for each performance characteristic. Instead, it presents study results and implies that meeting these results demonstrates substantial equivalence. For instance, in the method comparison, a Deming Regression with a slope close to 1 and an intercept close to 0, and a high correlation coefficient (R) suggests good agreement. Similarly, for other studies like precision, linearity, and interference, the reported results are expected to fall within acceptable ranges for diagnostic assays, which are generally implied by the use of CLSI guidelines.

Therefore, the table below will summarize the reported performance as presented in the document, rather than explicit pre-defined acceptance criteria.

Performance Characteristic	Reported Device Performance (LIAISON® XL 1,25 Dihydroxyvitamin D)	Predicate Device Performance (where applicable)
Method Comparison (vs. DiaSorin 1,25 Dihydroxyvitamin D 125I RIA)	Deming Regression: N=141, Slope = 0.973 (95% CI: 0.855 to 1.092), Intercept = -1.614 (95% CI: -7.475 to 4.248), R = 0.918	-
Sample Matrix Comparison (vs. Serum)	SST Serum: Slope = 1.011 (0.99 to 1.03), Intercept = -0.285 (-1.08 to 0.83), R² = 0.9908 EDTA Plasma: Slope = 1.010 (0.98 to 1.04), Intercept = 0.321 (-1.15 to 1.64), R² = 0.9975 Lithium Heparin: Slope = 1.0000 (0.97 to 1.04), Intercept = 0.1000 (-1.24 to 1.31), R² = 0.9957	-
Reference Range (U.S. Subjects, N=123)	Median = 47.8 pg/mL, Observed Range (2.5th to 97.5th Percentile) = 19.9 - 79.3 pg/mL	25.1 - 66.1 pg/mL (for predicate)
Precision (Total %CV across lots)	Kit Control 1: 3.8% Kit Control 2: 3.6% Prec Serum 1: 6.6% Prec Serum 2: 5.7% Prec Serum 3: 5.0% Prec Serum 4: 4.1% Prec Serum 5: 4.8% Prec Serum 6: 5.9% Cal Ver A: 5.7% Cal Ver B: 3.5% Cal Ver C: 3.6% Cal Ver D: 3.7%	-
Linearity (Dilution Linearity)	Observed 1,25 (OH)2 D = 0.981(Expected) + 0.005; R = 0.9994	-
High Dose Hook Effect	No hook effect observed up to 5000 pg/mL of 1,25 (OH)2 D	-
Recovery Study (Mean Recovery)	94%	-
Analytical Specificity (Cross-Reactivity)	1,25 (OH)2 D3: 103.4% 1,25 (OH)2 D2: 104.8% Zemplar: 113% 25(OH)D3: <0.1% (and other listed non-cross-reacting substances)	-
Interference Studies (Endogenous)	No significant interference (≤ ±10%) for: Hemoglobin (300 mg/dL), Bilirubin (40 mg/dL), Triglycerides (3,000 mg/dL), Cholesterol (400 mg/dL), Albumin (12 g/dL), Uric Acid (20 mg/dL), HAMA (3774 ng/mL), Rheumatoid Factor (7310 IU/mL)	-
Interference Studies (Exogenous)	No significant interference (≤ ±10%) for: Acetaminophen (20 mg/dL), Acetylsalicylic Acid (65 mg/dL), Salicylic Acid (60 mg/dL), Ibuprofen (50 mg/dL), Biotin (0.1mg/dL), Ascorbic Acid (6 mg/dL), Metaprolol (1.2 mg/dL), Propanolol (0.23 mg/dL), Hydrochlorothiazide (0.6 mg/dL), Furosemide (6 mg/dL), Valproic Acid (57.6 mg/dL), Spironolactone (0.6 µg/mL), Nifedipine (43 µg/dL), Verapamil (216 µg/dL), Losartan Potassium (2.25 µg/mL), Valsartan (11 µg/mL), Tetracycline (15.1 µg/mL), Enalapril (42.4 µg/dL), Doxycycline (34.6 µg/mL), Lisinopril (32.7 µg/dL)	-
Limit of Blank (LoB)	≤ 0.35 pg/mL	-
Limit of Detection (LoD)	0.70 pg/mL	-
Limit of Quantitation (LoQ)	5.0 pg/mL	-

2. Sample sized used for the test set and the data provenance (e.g. country of origin of the data, retrospective or prospective)

Method Comparison: 141 samples. The provenance of these samples (e.g., country of origin, retrospective/prospective) is not specified in the provided text.
Sample Matrix Comparison: 52 matched patient sets of serum, SST serum, EDTA plasma, and Lithium Heparin samples. Provenance not specified.
Reference Range: 123 apparently healthy adults aged 21-75 years from mixed ethnic backgrounds (48% dark skinned and 52% light skinned). Samples collected in winter (48.8%) and summer (51.2%) from subjects from the northern, central, and southern regions of the U.S. This is prospectively collected data from the U.S.
Precision: A coded panel of 6 frozen serum samples (2 spiked, 4 native), 2 lots of controls, and 2 lots of calibration verifiers. The origin of the serum samples is not specified.
Linearity (Dilution Linearity): One serum sample pool. Provenance not specified.
High Dose Hook Effect: A zero sample spiked with 1,25 (OH)2 D. This is a laboratory-prepared sample.
Recovery Study: Five (5) high concentration serum samples and 5 low concentration serum samples. Provenance not specified.
Analytical Specificity (Cross-Reactivity) & Interference Studies: Laboratory-prepared spiked samples and human serum samples. Provenance not specified for the base serum.
Limit of Blank, Limit of Detection, and Limit of Quantitation: Not specified, likely laboratory-generated data following CLSI guidelines.

3. Number of experts used to establish the ground truth for the test set and the qualifications of those experts (e.g. radiologist with 10 years of experience)

This document describes an in vitro diagnostic (IVD) immunoassay, not an imaging device or a device requiring human interpretation for ground truth establishment. Therefore, there are no experts used in the way described for establishing ground truth from image interpretation. The "ground truth" for this type of device is typically established through reference methods, gold standard assays (the predicate device in this case), and precise laboratory preparation of spiked or characterized samples.

4. Adjudication method (e.g. 2+1, 3+1, none) for the test set

Not applicable. This is an IVD immunoassay, and ground truth is based on quantitative measurements from established methods, not expert consensus or adjudication of interpretations.

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 an IVD immunoassay, not an imaging device or one involving human readers for interpretation.

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

Yes, the entire performance characterization presented in the document represents the standalone performance of the LIAISON® XL 1,25 Dihydroxyvitamin D assay. It's an automated chemiluminescent immunoassay, and its measurements are generated directly by the instrument, without a human-in-the-loop interpreting the results in a subjective manner as would be the case for an imaging algorithm. The output is a quantitative value of 1,25 dihydroxyvitamin D concentration.

7. The type of ground truth used (expert consensus, pathology, outcomes data, etc)

The "ground truth" for this assay is established in several ways:

Reference Method/Predicate Device: For method comparison, the DiaSorin 1,25 Dihydroxyvitamin D 125I RIA (K014030) served as the comparative method, acting as the gold standard for comparison.
Defined Concentrations/Spiking: For linearity, hook effect, recovery, cross-reactivity, and interference studies, samples were prepared with known, defined concentrations of the analyte or interfering/cross-reacting substances.
Healthy Population for Reference Range: For the reference range study, samples were collected from a defined "apparently healthy" adult population, with normal physiological values for related markers (Total Calcium, TSH, and PTH) to establish a normal range.
Internally Certified Standards: The calibrators, controls, and calibration verifiers are traceable to in-house standards prepared from certified reference material (1α, 25 dihydroxyvitamin D).

8. The sample size for the training set

This document describes the validation of an IVD assay, not a machine learning or AI algorithm in the typical sense that would have a "training set" for model development. The performance studies detailed are verification and validation studies for a laboratory assay. Therefore, the concept of a "training set" as understood in AI/ML is not directly applicable here. The assay's "learning" or optimization would have occurred during its development phase, preceding these regulatory submission studies, and would not have been for an "algorithm" as typically defined in AI.

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

As explained above, the concept of a "training set" as it pertains to AI/ML algorithms is not directly applicable to this IVD assay. The development process, which might be analogous to "training," would have involved optimizing assay reagents, conditions, and calibration procedures using various characterized samples and reference methods. The ground truth for such internal development would similarly rely on traceable standards, reference methods, and carefully prepared samples with known concentrations.

Summary

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December 10, 2014

Food and Drug Administration 10903 New Hampshire Avenue Document Control Center - WO66-G609 Silver Spring, MD 20993-0002

DIASORIN INC. CAROL DEPOUW REGULATORY AFFAIRS SPECIALIST 1951 NORTHWESTERN AVENUE STILLWATER MN 55082-0285

Re: K141463

Trade/Device Name: LIAISON® XL 1,25 Dihydroxyvitamin D; LIAISON® XL 1,25 Dihydroxyvitamin D Control Set; LIAISON® XL 1.25 Dihydroxyvitamin D Calibration Verifiers Regulation Number: 21 CFR 862.1825 Regulation Name: Vitamin D test system Regulatory Class: II Product Code: MRG, JJX Dated: November 12, 2014 Received: November 13, 2014

Dear Ms. Carol Depouw:

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 Parts 801 and 809); medical device reporting (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 Part 820); and if applicable, the

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electronic product radiation control provisions (Sections 531-542 of the Act); 21 CFR 1000-1050.

If you desire specific advice for your device on our labeling regulations (21 CFR Parts 801 and 809), please contact the Division of Industry and Consumer Education at its toll-free number (800) 638 2041 or (301) 796-7100 or at its Internet address

http://www.fda.gov/MedicalDevices/ResourcesforYou/Industry/default.htm. 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 the CDRH's Office of Surveillance and Biometrics/Division of Postmarket Surveillance.

You may obtain other general information on your responsibilities under the Act from the Division of Industry and Consumer Education at its toll-free number (800) 638-2041 or (301) 796-7100 or at its Internet address

http://www.fda.gov/MedicalDevices/ResourcesforYou/Industry/default.htm.

Sincerely yours,

Katherine Serrano -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

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Indications for Use

510(k) Number (if known) K141463

Device Name

LIAISON® XL 1,25 Dihydroxyvitamin D LIAISON® XL 1,25 Dihydroxyvitamin D Control Set LIAISON® XL 1,25 Dihydroxyvitamin D Calibration Verifiers

Indications for Use (Describe)

The LIAISON® XL 1,25 Dihydroxyviamin D Control Set is intended for use as assayed quality control samples to monitor the performance of the LIAISON® XL 1,25 Dihydroxyvitamin D assay.

The LIAISON® XL 1,25 Dihydroxyvitamin D Calibration Verifiers are assayed quality control materials intended for the quantitative verification of calibration and reportable range of the LIAISON® XL1,25 Dihydroxyvitamin D assay.

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)

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510(k) Summary

LIAISON® XL 1,25 Dihydroxyvitamin D LIAISON® XL 1,25 Dihydroxyvitamin D Control Set LIAISON® XL 1,25 Dihydroxyvitamin D Calibration Verifiers

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

1. 510(k) Number: K141463

1. Applicant: Carol A. DePouw DiaSorin Inc. 1951 Northwestern Avenue, Stillwater, MN 55082-0285 Office Number: 651-351-5850; Fax Number: 651-351-5669 Email: carol.depouw@diasorin.com

Date: May 30, 2014

4. Proprietary and Established Names:

LIAISON® XL 1,25 Dihydroxyvitamin D LIAISON® XL 1,25 Dihydroxyvitamin D Control Set LIAISON® XL 1,25 Dihydroxyvitamin D Calibration Verifiers

5. Requlatorv Information:

LIAISON® XL 1,25 Dihydroxyvitamin D

Requlation Section: 21 CFR 862.1825 Classification: Class II Product Code: MRG Panel: Clinical Chemistry (75)

LIAISON® XL 1,25 Dihydroxyvitamin D Control Set LIAISON® XL 1.25 Dihvdroxvvitamin D Calibration Verifiers

Requlation Section: 21 CFR 862.1660 Classification: Class I, reserved Product Code: JJX Panel: Clinical Chemistry (75)

6. Predicate Devices:

The predicate device used to demonstrate substantial equivalence to the LIAISON® XL 1,25 Dihydroxyvitamin D is the DiaSorin 1,25 Dihydroxyvitamin D 1251 RIA previously cleared under K014030.

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The predicate device used to demonstrate substantial equivalence to the LIAISON® XL 1,25 Dihydroxyvitamin D is the LIAISON® N-TACT® PTH Gen II Control Set previously cleared under K132515.

The predicate device used to demonstrate substantial equivalence to the LIAISON® XL 1,25 Dihydroxyvitamin D Calibration Verifiers is the LIAISON® N-TACT® PTH Gen II Calibration Verifiers previously cleared under K132515.

7. Device Description:

LIAISON® XL 1,25 Dihydroxyvitamin D Control set contains;

2 levels controls containing human serum spiked with 1,25 (OH)2 D, and . preservatives; 2 vials each level; lyophilized
The target concentration for control level 1 is 35 pg/mL. The target concentration for control Level 2 is 120 pg/mL.

The range of concentrations of each control is reported on the certificate of analysis provided with each LIAISON® XL 1.25 Dihydroxyvitamin D Control set.

LIAISON® XL 1,25 Dihydroxyvitamin D Calibration Verifier set contains:

4 levels containing human serum spiked with 1,25 (OH), D, and preservatives, . 1 vial each level, lyophilized
The target concentration for cal verifier A is 15 pg/mL. The target concentration for cal verifier B is 40 pg/mL. The target concentration for cal verifier C is 80 pg/mL. The target concentration for cal verifier D is 150 pg/mL.

The range of concentrations of each calibration verifier is reported on the certificate of analysis provided with each LIAISON® XL 1,25 Dihydroxyvitamin D Calibration Verifier set.

8. Intended Use:

The LIAISON® XL 1,25 Dihydroxyvitamin D is an in vitro chemiluminescent immunoassay (CLIA) intended for the quantitative determination of 1,25 dihydroxyvitamin (1,25(OH)» D) in serum, EDTA and Lithium Heparin plasma. Results

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of the 1,25 Dihydroxyvitamin D are used in the assessment of vitamin D sufficiency. Assay results should be used in conjunction with other clinical and laboratory data to assist the clinician in making individual patient management decisions in adult populations. The test is to be performed on the LIAISON® XL Analyzer.

The LIAISON® XL 1,25 Dihydroxyvitamin D Control Set is intended for use as assayed quality control samples to monitor the performance of the LIAISON® XL 1,25 Dihydroxyvitamin D assay.

9. Indication(s) for Use:

Same as Intended Use

10. Substantial Equivalence Information:

The LIAISON® XL 1,25 Dihydroxyvitamin D, LIAISON® XL 1,25 Dihydroxyvitamin D controls and LIAISON® XL 1,25 Dihydroxyvitamin D Calibration Verifiers are prepackaged reagents for use on an automated clinical chemistry analyzer. The DiaSorin 1,25 Dihydroxyvitamin D 1251 RIA consists of prepackaged reagents for nonautomated manual use. A comparison of the similarities and differences between the devices are provided in the following table:

Assay Similarities and Differences
Characteristic	New DeviceLIAISON® XL 1,25Dihydroxyvitamin D	Predicate Device1,25-Dihydroxyvitamin D 125I RIA(K014030)
Intended Use	For in vitro quantitative determinationof 1,25 Dihydroxyvitamin D	For in vitro quantitative determinationof 1,25 Dihydroxyvitamin D
Measured Analyte	1,25(OH)2 D	1,25(OH)2 D
Primary extraction	No	Yes
Calibration	Two-point calibration	Five-point calibration
Calibration interval	14 days	every run
Calibrators	2 levels	5 levels
Antibody	Mouse monoclonal	Rabbit polyclonal
Reagent Storage	in refrigerator @ 2-8°C	Same
Measuring range	5 – 200 pg/mL	5 – 200 pg/mL
Sample Matrix	Serum, SST serum, EDTA Plasma,and Lithium Heparin	Serum and EDTA Plasma
Sample size	75 μL	500 μL

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ManufacturersControls	2 levels	Same
Reference range	19.9 – 79.3 pg/mL	25.1 - 66.1 pg/mL

Control Similarities and Differences
Characteristic	New Device	Predicate Device
	LIAISON® XL 1,25 DihydroxyvitaminD Control Set	LIAISON® N-TACT® PTH Gen IIControl Set (K132515)
Intended Use	intended for use as assayed qualitycontrol samples to monitor theperformance of the LIAISON® XL1,25 Dihydroxyvitamin D	intended for use as assayed qualitycontrol samples to monitor theaccuracy and precision of theLIAISON®N-TACT® PTH Gen II
Storage	Store at 2-8°C until ready to use	Same
Levels	2 levels: lyophilizedLevel 1 (approx 35 pg/mL)Level 2 (approx 120 pg/mL)	2 levels: lyophilizedLevel 1 (approx 20 pg/mL)Level 2 (approx 30 pg/mL)

Calibration Verifiers Similarities and Differences
Characteristic	Candidate Device	Predicate Device
Characteristic	LIAISON® XL 1,25 Dihydroxyvitamin DCalibration Verifiers	LIAISON® N-TACT® PTH Gen IICalibration Verifiers (K132515)
Intended Use	assayed quality control materialsintended for the quantitativeverification of calibration andreportable range of the LIAISON®XL1,25 Dihydroxyvitamin D assay.	assayed quality control materialsintended for the quantitativeverification of calibration andreportable range of the LIAISON®N-TACT® PTH Gen II assay
Storage	2 to 8°C	Same
Levels	4 levels; lyophilizedCal Ver A (approx 15 pg/mL)Cal Ver B (approx 40 pg/mL)Cal Ver C (approx 80 pg/mL)Cal Ver D (approx 150 pg/mL)	4 levels; lyophilizedCal Ver A (approx 10 pg/mL)Cal Ver B (approx 150 pg/mL)Cal Ver C (approx 650 pg/mL)Cal Ver D (approx 1600 pg/mL)
Volume	2.0 mLs	Same

11. Standard/guidance Document Reference:

· CLSI Guideline EP5-A2, Evaluation of Precision Performance of Quantitative Measurement Methods;
CLSI Guideline EP6-A, Evaluation of Linearity of Quantitative Analytical Methods;
· CLSI Guideline EP7-A2, Interference Testing in Clinical Chemistry;
· CLSI Guideline EP09-A3 Measurement Procedure Comparison and Bias Estimation Using Patient Samples; Approved Guideline
CLSI Guideline EP17-A2 Evaluation of Detection Capability for Clinical Laboratory Measurement Procedures;

CLSI Guideline EP28-A3, Defining, Establishing and Verifying Reference Intervals in the Clinical Laboratory.

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12. Performance Characters:

Method Comparison

A method comparison study comparing the LIAISON® XL 1,25 Dihydroxyvitamin D assay to the DiaSorin 1.25-Dihydroxyvitamin D 1251 RIAassay was performed on 141 samples following CLSI EP9-A3.

Mean results for the 1,25-Dihydroxyvitamin D 1261 RIA assay and the singlicate results for LIAISON® XL 1,25 Dihydroxyvitamin D were plotted. Deming regression analyses were performed on the results across the range of the LIAISON® XL 1,25 Dihydroxyvitamin D assay.

Deming Regression
n	slope	95% CI	intercept	95% CI	R
141	0.973	0.855 to 1.092	-1.614	-7.475 to 4.248	0.918

Sample Matrix Comparison

Fifty-two (52) matched patient sets of serum, SST serum, EDTA plasma, and Lithium Heparin plasma samples were tested to determine if these sample types provide equivalent results on the LIAISON® XL 1,25 Dihydroxyvitamin D assay. The following results were obtained:

Serum vs.	Slope	95% CI	Intercept pg/mL	95% CI	R2
SST Serum	1.011	0.99 to 1.03	-0.285	-1.08 to 0.83	0.9908
EDTA Plasma	1.010	0.98 to 1.04	0.321	-1.15 to 1.64	0.9975
Lithium Heparin	1.0000	0.97 to 1.04	0.1000	-1.24 to 1.31	0.9957

Reference Range

It is recommended that each laboratory establish its own range of expected values.

To assess the expected reference range for the LIAISON® XL 1,25 Dihydroxyvitamin D a study was performed with serum samples from 123 apparently healthy adults aged 21 -75 years of age from mixed ethnic backgrounds (48% dark skinned and 52% light skinned). Samples were collected in the winter (48.8%) and summer (51.2%) from subjects with normal Total Calcium, TSH, and PTH values from the northern, central and southern regions of the U.S. Based on the 95% Reference Interval, the following values were established following CLSI guideline C28-A3.

U.S. Subjects	Median1,25 (OH)2 D	Observed Range2.5th to 97.5th Percentile
N=123	47.8 pg/mL	19.9 - 79.3 pg/mL

Precision

Precision testing was performed following CLSI Guideline EP5-A2, Evaluation of Precision Performance of Quantitative Measurement Methods; Approved Guideline -Second Edition.

A coded panel comprised of 6 frozen serum samples (2 spiked, 4 native samples) 2 lots of LIAISON® XL 1,25 Dihydroxyvitamin D controls (2 levels) and 2 lots of LIAISON® XL

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1,25 Dihydroxyvitamin D Calibration Verifiers were tested in the study. The precision panel samples and kit controls were tested on two lots of LIAISON® XL 1,25 Dihydroxyvitamin D in two replicates per run, 2 runs per day for 20 operating days for a total of 160 replicate results per sample. The 20 day results are summarized for the combined reagent lot numbers as sample mean 1,25 (OH)> D concentration in pg/mL, standard deviations and coefficient of variation (%CV) for Between lot and Total across lots.

Sample ID	n	mean(pg/mL)	Between-Lot		Total(Across Lots)
			SD	%CV	SD	%CV
Kit Control 1	160	30.9	0.84	2.7%	1.16	3.8%
Kit Control 2	160	122.9	6.09	5.0%	4.36	3.6%
Prec Serum 1	160	23.3	0.05	0.2%	1.53	6.6%
Prec Serum 2	160	38.9	0.63	1.6%	2.20	5.7%
Prec Serum 3	160	52.7	0.64	1.2%	2.65	5.0%
Prec Serum 4	160	76.0	1.33	1.7%	3.13	4.1%
Prec Serum 5	160	137.4	1.91	1.4%	6.55	4.8%
Prec Serum 6	160	193.4	5.53	2.9%	11.34	5.9%
Cal Ver A	160	13.4	0.36	2.7%	0.76	5.7%
Cal Ver B	160	38.9	2.76	7.1%	1.36	3.5%
Cal Ver C	160	78.4	3.37	4.3%	2.82	3.6%
Cal Ver D	160	153.0	1.35	0.9%	5.61	3.7%

The following results were obtained from 1 lot of kit controls and the same 6 serum samples with one kit lot assayed in duplicate in two assays per day over 20 operating days.

		Intra-Run		TOTAL (Within-lot)
Sample ID	mean	SD	%CV	SD	%CV
KC-1	30.3	0.75	2.5%	1.30	4.3%
KC-2	118.6	3.78	3.2%	4.15	3.5%
PREC-1	23.3	0.91	3.9%	1.81	7.8%
PREC-2	38.4	1.27	3.3%	2.58	6.7%
PREC-3	52.2	1.35	2.6%	2.79	5.3%
PREC-4	75.1	2.08	2.8%	3.40	4.5%
PREC-5	136.1	3.26	2.4%	6.91	5.1%
PREC-6	189.5	5.14	2.7%	11.09	5.9%

Linearity

Dilution Linearity:

One serum sample pool was diluted and analyzed by the LIAISON® XL 1,25 Dihydroxyvitamin D assay following CLSI EP6-A. The results were analyzed by regression of Observed 1,25 (OH)> D Concentration versus Expected 1,25 (OH)> D Concentration.

The resulting equation is: Observed 1,25 (OH)2 D = 0.981(Expected) + 0.005;R=0.9994

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High Dose Hook Effect

Testing was conducted to determine if the LIAISON® XL 1,25 Dihydroxyvitamin D assay is susceptible to artificially low results in the presence of very high levels of 1,25 (OH)2 D (Hook Effect). A zero sample was spiked with enough 1,25 (OH)2 D to equal concentrations above the assay measuring range of 200 pg/mL.

No hook effect was observed up to 5000 pg/mL of 1,25 (OH)2 D .

Recovery Study

Five (5) high concentration serum samples and 5 low concentration serum samples were analyzed neat on the LIAISON® XL 1,25 Dihydroxyvitamin D assay. Recovery samples were then prepared by mixing defined ratios of the high and low samples and tested in replicates of 5. The observed values were compared to the expected values to determine the % recovery.

Samples	DefinedConcentration	Expectedpg/mL	Observedpg/mL	%Recovery
High Sample 1 (HS1)	177.4
2 HS1 : 1 LS1		128.0	121.0	95%
1 HS1 : 1 LS1		102.6	97.3	95%
1 HS1 : 2 LS1		77.2	73.0	95%
Low Sample 1 (LS1)	27.8
High Sample 2 (HS2)	203.6
2 HS2 : 1 LS2		151.7	141.6	93%
1 HS2 : 1 LS2		125.0	115.4	92%
1 HS2 : 2 LS2		98.2	89.8	91%
Low Sample 2 (LS2)	46.3
High Sample 3 (HS3)	181.6
2 HS3 : 1 LS3		134.2	127.0	95%
1 HS3 : 1 LS3		109.8	101.8	93%
1 HS3 : 2 LS3		85.4	81.7	96%
Low Sample 3 (LS3)	38.0
High Sample 4 (HS4)	201.6
2 HS4 : 1 LS4		143.0	137.6	96%
1 HS4 : 1 LS4		112.9	105.4	93%
1 HS4 : 2 LS4		82.7	77.4	94%
Low Sample 4 (LS4)	24.2
High Sample 5 (HS5)	223.8
2 HS5 : 1 LS5		158.3	148.4	94%
1 HS5 : 1 LS5		124.6	111.8	90%
1 HS5 : 2 LS5		90.8	85.1	94%
Low Sample 5 (LS5)	25.3
			Mean Recovery	94%

Analytical Specificity

Cross-Reactivity Studies

CLSI Guideline EP7-A2, Interference Testing in Clinical Chemistry; Approved Guideline; Second Edition.

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Controlled studies of potentially cross reacting substances were performed on the LIAISON® XL 1,25 Dihydroxyvitamin D assay at the concentrations listed below.

Cross-Reactant	SpikedConcentration	% CrossReactivity
1,25 (OH)2 D3	50 pg/mL	103.4%
1,25 (OH)2 D2	50 pg/mL	104.8%
Zemplar	100 pg/mL	113%
25(OH)D3	100,000 pg/mL	<0.1%
25(OH)D2	50,000 pg/mL	<0.1%
24,25 (OH)2 D3	50,000 pg/mL	<0.1%
25,26 (OH)2 D3	50,000 pg/mL	<0.1%
3-epi 25 (OH)D3	100,000 pg/mL	<0.1%
Vitamin D2	50,000 pg/mL	<0.1%
Vitamin D3	50,000 pg/mL	<0.1%
Sensipar	220,000 pg/mL	<0.1%

Interference Studies

Controlled studies of potentially interfering endogenous substances performed in serum with 1,25 (OH)> D levels up to 60 pg/mL showed no interference in the LIAISON® XL 1,25 Dihydroxyvitamin D at the highest concentration for each substance listed below.

Drug/Substance	Highest Concentration atwhich no significantinterference(≤ ±10%) was observed
Hemoglobin	300 mg/dL
Bilirubin (conjugated)	40 mg/dL
Bilirubin (unconjugated)	40 mg/dL
Triglycerides	3,000 mg/dL
Cholesterol	400 mg/dL
Albumin	12 g/dL
Uric Acid	20 mg/dL
HAMA	3774 ng/mL
Rheumatoid Factor	7310 IU/mL

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Controlled studies of potentially interfering exogenous substances performed in serum with 1,25 (OH)2 D levels up to 60 pg/mL showed no interference in the LIAISON® XL 1,25 Dihydroxyvitamin D at the highest concentration for each substance listed below.

Drug/Substance	Highest Concentration atwhich no significantinterference (≤ ±10%) wasobserved.
Acetaminophen	20 mg/dL
Acetylsalicylic Acid	65 mg/dL
Salicylic Acid	60 mg/dL
Ibuprofen	50 mg/dL
Biotin	0.1mg/dL
Ascorbic Acid	6 mg/dL
Metaprolol	1.2 mg/dL
Propanolol	0.23 mg/dL
Hydrochlorothiazide	0.6 mg/dL
Furosemide	6 mg/dL
Valproic Acid	57.6 mg/dL
Spironolactone	0.6 µg/mL
Nifedipine	43 µg/dL
Verapamil	216 µg/dL
Losartan Potassium	2.25 µg/mL
Valsartan	11 µg/mL
Tetracycline	15.1 µg/mL
Enalapril	42.4 µg/dL
Doxycycline	34.6 µg/mL
Lisinopril	32.7 µg/dL

Limit of Blank, Limit of Detection and Limit of Quantitation

The Limit of Blank, Limit of Detection and Limit of Quantitation were determined according to CLSI EP17-A2: Evaluation of Detection Capability for Clinical Laboratory Measurement Procedures; Approved Guideline June 2012 - Second Edition.

The following limits were determined with the LIAISON® XL 1,25 Dihydroxyvitamin D Assay:

LoB	LoD	LoQ
≤ 0.35 pg/mL	0.70 pg/mL	5.0 pg/mL

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Product	Storage Conditions		Claimed stability
Reagent Integral	Open vial	at 2-8°C	28 days
Binding Agent	Open vial	at 2-8°C	2 days
Calibrators	Open vial -Reconstituted	On system/Room temp	6 hours
	Open vial -Reconstituted	2-8°C	14 days
Calibration curve	N/A	N/A	14 days
Controls	Open vial -Reconstituted	On system/Room temp	6 hours
	Open vial -Reconstituted	2-8°C	28 days
Calibration Verifiers	Open vial -Reconstituted	On system/Room temp	6 hours
	Open vial -Reconstituted	2-8°C	28 days

Traceability

The LIAISON® XL 1,25 Dihydroxyvitamin D Calibrators, Controls and Calibration Verifiers are traceable to in-house standards prepared from certified reference material 1α. 25 dihvdroxvvitamin D.

Value Assignment

Calibrators

A minimum of 5 vials of each level of calibrator are tested on a minimum of 3 LIAISON® XL Analyzers, in a minimum of 5 assay runs with six replicates per vial resulting in a minimum of 30 individual replicate results per calibrator level for final value assignment.

Controls

A minimum of 10 vials of each level of control are tested on 2 different LIAISON® XL 1.25 Dihvdroxvvitamin D assay kit lots on a minimum of 3 LIAISON® XL Analyzers, in a minimum of 5 assay runs with 4 replicates per vial resulting in a minimum of 40 individual replicate results per control level for final value assignment. Final control ranges are established based on ± 2 Standard Deviations.

Calibration Verifiers

A minimum of 10 vials of each level of calibration verifier are tested on 2 different LIAISON® XL 1,25 Dihydroxyvitamin D assay kit lots on a minimum of 3 LIAISON® XL Analyzers, in a minimum of 5 assay runs with 4 replicates per vial resulting in a minimum of 40 individual replicate results per level for final value assignment. Final calibration verifier ranges are established based on ± 2 Standard Deviations.

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13. Conclusion:

The LIAISON®XL 1,25 Dihydroxyvitamin D is substantially equivalent in principle and
performance to the DiaSorin 1,25 Dihydroxyvitamin D 12°1 RIA. Accuracy was demonstrated by a Method Comparison.

The LIAISON® XL 1,25 Dihydroxyvitamin D Control Set and the LIAISON® XL 1,25 Dihydroxyvitamin D Calibration Verifiers are substantially equivalent in principle and performance to the LIAISON® N-TACT® PTH Gen II Control Set and LIAISON® N-TACT® PTH Gen II Calibration Verifiers, respectively.

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.