ALFIS Vitamin D in conjunction with ALFIS-3 Analyzer is an enzyme-linked fluorescence immunoassay intended for in vitro diagnostic use at clinical laboratories for the quantitative measurement of Total 25-hydroxy Vitamin D (25-OH Vitamin D) in human serum, lithium heparin plasma and sodium heparin plasma.

ALFIS Vitamin D is indicated to be used as an aid in the determination of Vitamin D sufficiency in adults.

ALFIS-3 Analyzer is a fluorescence-scanning instrument using magnetic beads and alkaline phosphatase enzyme system for in vitro diagnostic use in conjunction with various ALFIS immunoassays intended for measuring the concentration of designated analytes in human blood and other specimens.

Device Description

ALFIS Vitamin D Test Cartridge is a plastic structure molded in the form of a disposable, self-contained, unitized device which houses the 'magnetic bead', 'antibody-alkaline phosphatase-conjugator (Ab-ALP)', 'sample diluent', 'diethanolamine (DEA)', '4-Methylumbelliferyl phosphate (MUP)', 'washing buffer'; all of which are integral components of ALFIS Vitamin D test.

ALFIS Vitamin D test cartridge is an elongated structure having 150.8 mm length, 17 mm width and 16 mm height.

'ALFIS Vitamin D Test ID Chip' is a flat, rectangular device with its main body measuring 23 mm × 27 mm. Half of the portion along the breadth of the main body is 5 mm thick while remaining half is 3 mm in thickness. Another rectangular portion measuring 12 mm × 10 mm × 2 mm protrudes out from the breadth of apical side of the 3 mm-thick portion of the main body.

ALFIS Vitamin D Test ID Chip is an electronic memory device fitted into a plastic matrix. Lot-specific 'ALFIS Vitamin D Test ID Chip' is an integral component of ALFIS Vitamin D test system.

ALFIS-3 analyzer is a compact, bench-top, automated, fluorometric analyzer measuring 422 mm (L) x 270 mm (W) x 292 mm (H). ALFIS-3 weighs 13.0 kg.

ALFIS-3 analyzer is a fluorometer instrument of closed-system analyzer type.

'ALFIS Vitamin D Calibrators' needs to be tested by user laboratories for periodic calibration of ALFIS Vitamin D test system.

'ALFIS Vitamin D Controls' needs to be tested by user laboratories periodically for monitoring the performance of ALFIS Vitamin D test system.

AI/ML Overview

The provided text describes the performance evaluation studies for the ALFIS Vitamin D and ALFIS-3 Analyzer system, which is an in vitro diagnostic device for quantitative measurement of Total 25-hydroxy Vitamin D.

Here's a breakdown of the requested information based on the provided document:

Acceptance Criteria and Device Performance

The document doesn't explicitly state "acceptance criteria" in a tabular format with specific numerical targets for each parameter (e.g., "LoD < X ng/mL"). Instead, it describes how each performance characteristic was evaluated and the resulting performance data, often concluding that the results are "acceptable" or "insignificant interference." However, we can infer the acceptance for some parameters from the reported findings. For instance, for linearity, the high R² value (0.9998) demonstrates acceptable linearity. For precision (repeatability and reproducibility), the CV < 10% is explicitly stated as indicating acceptability. For LoQ, the target of CV ≤ 20% is mentioned.

Here's a table summarizing the reported device performance, with inferred acceptance criteria where explicit values are not provided as a target but rather indirectly through the statement of acceptability:

Performance Characteristic	Acceptance Criteria (Inferred/Stated)	Reported Device Performance
Limit of Blank (LoB)	(Implied to be low enough for accurate measurement)	1.82 ng/mL
Limit of Detection (LoD)	(Implied to be low enough for accurate measurement)	3.76 ng/mL
Limit of Quantitation (LoQ)	Inter-assay CV ≤ 20%	6.0 ng/mL (with CV ≤ 20% met)
Linearity	High correlation (R²) over claimed measuring range (6-100 ng/mL)	y = 1.0708x - 0.0757, R² = 0.9998 (over 6-100 ng/mL)
High-dose Hook Effect	No hook/prozone effect up to 1000 ng/mL	No hook/prozone effect observed up to 1000 ng/mL
Analytical Specificity (Interference)	Analyte recovery within 90-110% in presence of interferents	Performance not significantly affected; analyte recovery 90-110% for tested interferents
Analytical Specificity (Cross-reactivity)	Acceptable cross-reactivity with structural analogs	Detailed percentage cross-reactivity reported for various analogs (e.g., 99.30-100.61% for 25-hydroxyvitamin D3)
Repeatability (In-house)	CV < 10%	CV < 10%
Reproducibility (In-house)	CV < 10%	CV < 10%
Site-to-Site Reproducibility	CV < 10%	CV < 10%
Matrix Comparison	Acceptable commutability across matrices (Serum, Li-heparin, Na-heparin plasma)	Serum vs. Li-heparin Plasma: y=0.9797x+0.3501, R=0.9982. Serum vs. Na-heparin Plasma: y=0.98x+0.3486, R=0.9981.
Method Comparison (vs. RMP)	Acceptable compatibility/correlation with RMP	y = 0.997x + 0.4725, R=0.978 (for 6.45 - 83.33 ng/mL)
Method Comparison (vs. Predicate)	Acceptable compatibility/correlation with predicate device	y = 1.006x + 0.05392, R=0.9844 (for 6.18 - 79.48 ng/mL)

Study Details

Here's the detailed information regarding the studies:

A table of acceptance criteria and the reported device performance: (Refer to the table above).
Sample sizes used for the test set and the data provenance:
- Limit of Blank (LoB): 72 blank replicates per lot/analyzer pair (total 3 lots, 3 analyzers) tested across 3 days. This implies 72 * 3 = 216 replicates in total.
- Limit of Detection (LoD): 15 replicates per lot/analyzer pair (total 3 lots, 3 analyzers) for each of 4 low 25-hydroxyvitamin D-spiked serum samples. Total 45 * 4 = 180 replicates for samples (plus an unstated number for blanks).
- Limit of Quantitation (LoQ): 12 replicates per lot/analyzer pair (total 3 lots, 3 analyzers) for each of 5 low 25-hydroxyvitamin D serum samples. Total 36 * 5 = 180 replicates for samples.
- Linearity: 19 25-hydroxyvitamin D-spiked serum samples, tested in triplicate. Total 19 * 3 = 57 measurements.
- High-dose Hook Effect: A series of spiked samples (10 concentrations from 12.5 ng/mL to 1000 ng/mL), tested in triplicate. Total 10 * 3 = 30 measurements.
- Analytical Specificity (Interference): The specific number of samples for each interferent is not explicitly stated (e.g., "Performance... not been found to be significantly affected when the endogenous and exogenous substances were tested at specified highest concentrations").
- Analytical Specificity (Cross-reactivity): The specific number of samples for each cross-reactant is not explicitly stated.
- In-house repeatability and reproducibility:
  - Repeatability: 3 spiked serum samples, tested in duplicate daily for 20 successive days. Total 3 * 2 * 20 = 120 measurements.
  - Reproducibility: 3 spiked serum samples, tested in quintuplicate daily for 5 successive days (with 3 cartridge lots/analyzer/operator pairs). Total 3 * 5 * 5 = 75 measurements per lot/analyzer/operator pair for reproducibility (75 * 3 = 225 total).
- External site-to-site reproducibility: 3 spiked serum samples, tested in quintuplicate daily for 5 successive days (across 3 sites/cartridge lots/analyzer-operators). Total 3 * 5 * 5 = 75 measurements per site, so 75 * 3 = 225 in total.
- Matrix Comparison: 45 matching clinical serum, lithium heparin plasma, and sodium heparin plasma samples, plus an additional 5 spiked samples. Total 45 + 5 = 50 samples per matrix type.
- Reference Interval: Serum samples collected from 299 apparently healthy adults in the United States. Data provenance is specified as US (central, northern, southern states), collected from external clinical sample repositories.
- Method Comparison Study: 120 CDC VDSCP verification samples. Provenance of these samples is implied as US (CDC samples). The study was carried out at the "in-house laboratory."
Data Provenance (Retrospective/Prospective): The document does not explicitly state whether the samples were collected retrospectively or prospectively for most studies. However, for the "Reference Interval" study, it mentions "samples collected from 299 apparently healthy adults in the United States," and "sample sets procured... met the following criteria: ...Collected after June 2021", which suggests recent, potentially prospective, collection or procurement for the study. For the method comparison, CDC VDSCP verification samples are typically existing, well-characterized samples, suggesting a retrospective use of these samples for the study.
Number of experts used to establish the ground truth for the test set and the qualifications of those experts: This information is not provided. For this type of IVD device (quantitative measurement of a biomarker), "ground truth" is typically established by certified reference methods, not human expert interpretation in the way it would be for an imaging AI device.
Adjudication method (e.g. 2+1, 3+1, none) for the test set: Not applicable for this type of quantitative IVD device. Ground truth is established by analytical methods or reference measurement procedures (RMP).
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 device is an automated immunoassay system, not an AI-assisted diagnostic tool requiring human reader studies or MRMC.
If a standalone (i.e. algorithm only without human-in-the-loop performance) was done: The device itself is an automated, standalone system (ALFIS-3 Analyzer in conjunction with ALFIS Vitamin D cartridges) designed to measure 25-OH Vitamin D quantitatively without direct human interpretation of a visual output. The device results are numerical values. The performance studies (LoB, LoD, LoQ, Linearity, Precision, Analytical Specificity, Matrix Comparison, Method Comparison) all represent the "standalone" performance of the system.
The type of ground truth used (expert consensus, pathology, outcomes data, etc):
- For LoB, LoD, LoQ, Linearity, High-dose Hook, Analytical Specificity, Repeatability, Reproducibility, Matrix Comparison: Ground truth is based on the known concentrations of spiked samples or reference materials, or properties of the substances themselves.
- For Method Comparison: Ground truth for the samples was established by the Reference Measurement Procedure (RMP) of 25-hydroxyvitamin D, i.e., ID-LC-MS/MS at a CDC certified laboratory with NIST standard reference material 2972. This is a highly robust and accepted method for establishing ground truth in clinical chemistry.
The sample size for the training set: Not applicable for this type of immunoassay device. The device operates based on a pre-defined enzyme-linked fluorescence immunoassay protocol, not a machine learning algorithm that requires a "training set" in the computational sense. The "training" for the device would involve calibration using provided calibrators.
How the ground truth for the training set was established: Not applicable. The "training set" concept doesn't apply here. However, for calibration, the instructions state that "ALFIS Vitamin D Calibrators" are used, with approximate assigned 25-OH Vitamin D levels (10 ng/mL and 40 ng/mL), and that a "Lot-specific master calibration curve/equation is encoded in the 'Lot-specific ALFIS Vitamin D Test ID Chip'." This implies that the calibrators' "ground truth" levels (or reference values) are pre-established by the manufacturer, likely traceable to a recognized standard such as the CDC RMP.

Summary

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September 22, 2023

Immunostics Inc. % Parag Bhurchandi Principal Research Scientist (Regulatory Affairs) Boditech Med Inc. 43, Geodudanji 1-gil, Dongnae-myeon, Chuncheon-si, Gang-won-do 24398 Korea. South

Re: K221817

Trade/Device Name: ALFIS Vitamin D, ALFIS-3 Analyzer Regulation Number: 21 CFR 862.1825 Regulation Name: Vitamin D Test System Regulatory Class: Class II Product Code: MRG, KHO Dated: March 24, 2023 Received: March 27, 2023

Dear Parag Bhurchandi:

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. Although this letter refers to your product as a device, please be aware that some cleared products may instead be combination products. The 510(k) Premarket Notification Database located at https://www.accessdata.fda.gov/scripts/cdrh/cfdocs/cfpmn/pmn.cfm identifies combination product submissions. 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.

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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) for devices or postmarketing safety reporting (21 CFR 4, Subpart B) for combination products (see https://www.fda.gov/combination-products/guidance-regulatory-information/postmarketing-safety-reportingcombination-products); good manufacturing practice requirements as set forth in the quality systems (QS) regulation (21 CFR Part 820) for devices or current good manufacturing practices (21 CFR 4, Subpart A) for combination products; 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 https://www.fda.gov/medical-device-safety/medical-device-reportingmdr-how-report-medical-device-problems.

For comprehensive regulatory information about mediation-emitting products, including information about labeling regulations, please see Device Advice (https://www.fda.gov/medicaldevices/device-advice-comprehensive-regulatory-assistance) and CDRH Learn (https://www.fda.gov/training-and-continuing-education/cdrh-learn). 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 (https://www.fda.gov/medical-device-advice-comprehensive-regulatoryassistance/contact-us-division-industry-and-consumer-education-dice) for more information or contact DICE by email (DICE@fda.hhs.gov) or phone (1-800-638-2041 or 301-796-7100).

Sincerely.

Marianela Perez-torres -S

Marianela Perez-Torres, Ph.D. Acting Director Division of Chemistry and Toxicology Devices OHT7: Office of In Vitro Diagnostics Office of Product Evaluation and Ouality Center for Devices and Radiological Health

Enclosure

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Indications for Use

510(k) Number (if known) K221817

Device Name

ALFIS Vitamin D. ALFIS-3 Analyzer

Indications for Use (Describe)

ALFIS Vitamin D is indicated to be used as an aid in the determination of Vitamin D sufficiency in adults.

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

X Prescription Use (Part 21 CFR 801 Subpart D)

| Over-The-Counter Use (21 CFR 801 Subpart C)

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Section 5: 510(k) Summary

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

Device Type/Common Name: Radioimmunoassay, Vitamin D Test System

Trade/Proprietary Name	: ALFIS Vitamin DALFIS-3 Analyzer
Type of Submission	: Traditional 510(k)
Basis for Submission	: Traditional 510(k) Submission for a New Device
Submitter/Applicant/Sponsor	: Immunostics Inc.38 Industrial Way E,Eatontown, New Jersey, 07724,United StatesPhone No.: 732-918-0770Fax No.: 732-918-0618Website: www.immunostics.com
Applicant Contact Person	: Dr. Young Mi KimDirector (RA & QA)Phone No.: 732-918-0770Fax No.: 732-918-0618E-mail: ymkim@immunostics.com
Official correspondent	: Dr. Parag BhurchandiPrincipal Research Scientist (Regulatory Affairs)Boditech Med. Inc.43, Geodudanji 1-gil, Dongnae-myeon,Chuncheon-si, Gangwon-do 24398,Republic of Korea (South Korea)Phone No.: +82 33 243 1435Fax No.: +82 33 243 9373E-mail: parag@boditech.co.kr
Classification Regulation	: 21CFR § 862.1825 Vitamin D test system
Class	: II (for ALFIS Vitamin D Test Cartridges)I (for ALFIS-3 Analyzer)
Panel	: Clinical Chemistry
Product Code	: MRG; Vitamin D Test System

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KHO; Fluorometer for Clinical Use

Predicate Device	: Elecsys Vitamin D Total III
	with cobas e 601 analzyer
	Roche Diagnostics GMBH
	Sandhofer Strasse 116
	Mannheim Baden-Wurttenberg,
	DE D-68305

Predicate Device 'K' Number : K210901

Predicate Device Product Code: MRG, Vitamin D Test System

Intended use of ALFIS Vitamin D:

Indications for use of ALFIS Vitamin D:

ALFIS Vitamin D is indicated to be used as an aid in the determination of Vitamin D sufficiency in adults.

Intended use/indication for use of ALFIS-3 Analyzer:

Description of ALFIS Vitamin D Test Cartridges:

ALFIS Vitamin D test cartridge is an elongated structure having 150.8 mm length, 17 mm width and 16 mm height.

Description of ALFIS Vitamin D Test ID Chip:

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Another rectangular portion measuring 12 mm × 10 mm × 2 mm protrudes out from the breadth of apical side of the 3 mm-thick portion of the main body.

ALFIS Vitamin D Test ID Chip is an electronic memory device fitted into a plastic matrix. Lot-specific 'ALFIS Vitamin D Test ID Chip' is an integral component of ALFIS Vitamin D test system.

Before initiating the test run for testing a clinical sample or calibrator/control using ALFIS Vitamin D Test Cartridge belonging to a new lot for the first time, the operator needs to mandatorily insert the lot-specific ALFIS Vitamin D Test ID Chip into the 'ID Chip Port' of the ALFIS-3 analyzer.

Description of ALFIS-3 Analyzer:

For performing ALFIS Vitamin D test on a clinical sample or an ALFIS Vitamin D Control, ALFIS Vitamin D test cartridge needs to be used in conjunction with the ALFIS-3 analyzer.

ALFIS-3 analyzer is a compact, bench-top, automated, fluorometric analyzer measuring 422 mm (L) x 270 mm (W) x 292 mm (H). ALFIS-3 weighs 13.0 kg.

ALFIS-3 analyzer is a fluorometer instrument of closed-system analyzer type. The operator does not have access to those configuration parameters that could affect the assay process, test analysis or result calculation or any other parameter that could affect test result outcome. These and other operational parameters can only be configured by the 'System Administrator' through software and/or firmware upgrade and modification of operational settings in consultation with the manufacturer or an authorized service provider.

With a simple computerized touchscreen user interface, the analyzer enables the operator to initiate the test run which is completed automatically and terminates with display of test result or an error message alert (in case of any procedural error and/or system malfunction).

Description of ALFIS Vitamin D Calibrators:

'ALFIS Vitamin D Calibrators' needs to be tested by user laboratories for periodic calibration of ALFIS Vitamin D test system.

Considering the reportable range of ALFIS Vitamin D test (6-100 ng/mL), the sponsor provides ALFIS Vitamin D Calibrators with following approximate assigned 25-OH Vitamin D levels:

No.	ALFIS Vitamin D Calibrator	Approximate Assigned/Target 25(OH)D Level
1	ALFIS Vitamin D Calibrator Level 1	10 ng/mL
2	ALFIS Vitamin D Calibrator Level 2	40 ng/mL

Analyte levels of ALFIS Vitamin D Calibrators

The sponsor would market a set of 'ALFIS Vitamin D Calibrator Level 1 and Level 2' separately from the ALFIS Vitamin D test product on demand by the user laboratories.

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Description of ALFIS Vitamin D Controls:

'ALFIS Vitamin D Controls' needs to be tested by user laboratories periodically for monitoring the performance of ALFIS Vitamin D test system.

Considering the reportable range (6 - 100 ng/mL) of ALFIS Vitamin D and usual adult reference intervals of 25(OH)D IVD tests, the sponsor provides ALFIS Vitamin D Controls with following approximate assigned 25-OH Vitamin D levels:

No.	ALFIS Vitamin DControl	Approximate Assigned/Target 25(OH)DLevel	AcceptableRange
1	ALFIS Vitamin DControl Level 1	15 ng/mL	13.5 ~ 16.5 ng/mL
2	ALFIS Vitamin DControl Level 2	35 ng/mL	31.5~38.5 ng/mL

Analyte levels and reference ranges of ALFIS Vitamin D Controls

The sponsor would market a set of 'ALFIS Vitamin D Control Level 1 and Level 2' separately from the ALFIS Vitamin D test product on demand by the user laboratories.

Similarities between ALFIS Vitamin D (with ALFIS-3 analyzer) and the predicate device Elecsys Vitamin D Total III:

Following table shows the similarities between ALFIS Vitamin D (with ALFIS-3 analyzer) and the predicate device Elecsys Vitamin D Total III

Table 5.1: Similarities between ALFIS Vitamin D (with ALFIS-3 analyzer)and the predicate device Elecsys Vitamin D Total III
No.	Comparison Parameter	Candidate Device/TestALFIS Vitamin D(with ALFIS-3 Analyzer)	Elecsys Vitamin DTotal III
1	Nature of the test	Quantitative in vitro diagnostictest system for total 25-hydroxyvitamin D	Same
2	Indications for use(s)	As an aid in the assessment ofVitamin D sufficiency in adults	Same
3	Intended use sites	Clinical laboratories	Same
4	Sample type	Serum or plasma	Same
5	Result unit	ng/mL	Same
No.	ComparisonParameter	Candidate Device/TestALFIS Vitamin D(with ALFIS-3)	Predicate Device/TestElecsys Vitamin D Total III
Table 5.2: Differences between ALFIS Vitamin D (with ALFIS-3) and the predicate device Elecsys Vitamin D Total III
1	Detectionprotocol	Enzyme-linked fluorescence assay	Electrochemiluminescence
2	Measuringrange	6-100 ng/mL	6-125 ng/mL6-240 ng/mL (after dilution)
3	Associatedinstrument	ALFIS-3 Analyzer	Cobas e analyzers
4	Test format	Self-contained, unitized, ready-to-use, disposable cartridge to be usedin conjunction with ALFIS-3analyser	A kit for multiple use with threeready to use liquid assay reagentsand two pretreatment reagents;capable of testing up to 43 samplesat a time
5	Turnaroundtime	Total turnaround time = 36 minutes	Total duration of assay = 27minutes
6	Sample volumerequired per test	100 μL	20 μL
7	Sample volumeutilized per test	40 μL	20 μL
8	Limit ofQuantitation/FunctionalSensitivity	6.0 ng/mL(with imprecision of CV ≤ 20%)	6.0 ng/mL(with imprecision of CV ≤ 20%)
9	Calibration	Lot-specific master calibrationcurve/equation is encoded in the'Lot-specific ALFIS Vitamin D TestID Chip' which needs to be insertedin the 'ID Chip Port' of ALFIS-3analyzer prior to using that ALFISVitamin D test cartridge lot for thefirst time.User-level periodic calibration tomodify the pre-defined mastercalibration curve (encoded in the IDchip) needs to be performed by	Every Elecsys reagent set has abarcoded label containing specificinformation for calibration of theparticular reagent lot.The predefined master curve isadapted to the analyzer using therelevant calibrators.

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Differences between ALFIS Vitamin D (with ALFIS-3 Analyzer) and the predicate device Elecsys Vitamin D Total III.

Following table shows the differences between ALFIS Vitamin D (with ALFIS-3 Analyzer) and the predicate device Elecsys Vitamin D Total III.

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testing ALFIS Vitamin D Calibrators
Level 1 and 2 with the ALFIS
Vitamin D test system.

Summary of Performance Evaluation Studies:

1) Limit of Blank:

Limit of Blank (LoB) of ALFIS Vitamin D test has been evaluated as per CLSI EP -17-A2.
-6 replicates of 4 blank (25-hydroxyvitamin D-depleted) serum samples were tested with 3 lots of test cartridges on 3 ALFIS-3 analyzers (1 lot/analyzer) for 3 successive days; thus testing 72 blank replicates per lot/analyzer pair.
-Limit of Blank (LoB) was calculated by non-parametric analysis of the data.
-Limit of Blank of ALFIS Vitamin D test is 1.82 ng/mL.

2) Limit of Detection:

-Limit of Detection (LoD) of ALFIS Vitamin D test has been evaluated as per CLSI EP 17-A2.
5 replicates each of 4 low 25-hydroxyvitamin D-spiked serum samples were tested with 3 lots of test cartridges on 3 respective analyzers (1 lot/analyzer) for 3 successive days; thus testing 15 replicates per lot/analyzer pair and total 45 replicates per sample.
Limit of Detection (LoD) was calculated by parametric analysis of the data. -
-Limit of Detection of ALFIS Vitamin D test is 3.76 ng/mL.

3) Limit of Quantitation:

Limit of Quantitation (LoQ) of ALFIS Vitamin D has been evaluated as per CLSI EP -17-A2.
4 replicates of each of the 5 low 25-hydroxyvitamin D serum samples were tested with 3 lots of test cartridges on 3 respective analyzers (1 lot/analyzer) for 3 successive days; thus testing 12 replicates per lot/analyzer pair and total 36 replicates per sample.
-Limit of Quantitation (LoQ) was calculated by considering accuracy goal of interassay CV ≤ 20%.
-Limit of Quantitation of ALFIS Vitamin D test is 6.0 ng/mL

4) Linearity and Reportable Range:

For evaluating linearity and measuring/reportable range of ALFIS Vitamin D test, a series of 19 25-hydroxyvitamin D-spiked serum samples spanning the concentration

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range of 1.3 ng/mL - 130 ng/mL was tested in triplicate with the one lot of ALFIS Vitamin D test cartridges on one ALFIS-3 analyzer on the same day.

Mean of the triplicate test results of each sample was plotted against its expected TSH concentration calculated mathematically.
ALFIS Vitamin D test showed linearity over the entire claimed measuring/reportable range of 6-100 ng/mL with following linear regression equations.

y = 1.0708x - 0.0757, R2= 0.9998

5) Susceptibility to High-dose Hook Effect:

For evaluating susceptibility to high dose hook (prozone) effect, a series of spiked samples having approximate 25-hydroxyvitamin D concentrations 12.5 ng/mL, 25 ng/mL, 50 ng/mL, 75 ng/mL, 100 ng/mL, 150 ng/mL, 200 ng/mL, 500 ng/mL, 750, and 1000 ng/mL were tested in triplicate with one lot of test cartridges using one analyzer on the same day by the same operator.
-No hook/prozone effect was observed up to 25-hydroxyvitamin D concentration of 1000 ng/mL.

6) Analytical specificity:

트 Analytical specificity of ALFIS Vitamin D has been evaluated as per CLSI EP07, 3rd edition.

Interference from endogenous and exogenous substances:

Susceptibility of ALFIS Vitamin D test to interference from various endogenous . and exogenous substances has been evaluated.
Performance of ALFIS Vitamin D test has not been found to be significantly 트 affected when the endogenous and exogenous substances were tested at specified highest concentrations as follows.
. ALFIS Vitamin D test showed analyte recovery within the acceptable range of 90-110% in presence of specified concentrations of above endogenous and exogenous interferants thereby indicating insignificant interference.

Potential endogenous interferent tested	Highest concentration tested at which nosignificant interference was observed
Albumin/Protein	12,000 mg/dL
Conjugated Bilirubin	40 mg/dL
Human hemoglobin	1,000 mg/dL
Triglycerides	2,000 mg/dL
HAMA	800 ng/mL
Rheumatoid Factor	200 IU/mL
Biotin	3,500 ng/mL

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Potential exogenous interferent tested	Highest concentration tested at which nosignificant interference was observed
Acetaminopen	15.5 mg/dL
Acetylcysteine	15 mg/dL
Acetylsalicylic acid	3.0 mg/dL
Ampicillin-Na	7.5 mg/dL
Cyclosporin	180 µg/dL
Doxycycline	1.8 mg/dL
Ibuprofen	22 mg/dL
Itraconazole	3.0 mg/dL
Levodopa	750 µg/dL
Methyldopa	2.25 mg/dL
Rifampicin	4.8 mg/dL
Ascorbic acid	5.25 mg/dL

Cross-reactivity with structural analogs of 25-hydroxyvitamin D:

. Cross-reactivity of ALFIS Vitamin D test system with various structural analogs of 25-hydroxyvitamin D has been evaluated.
. The cross-reactivity was determined using the following formula:

% cross reactivity = [(mean recovery of test samples) - (mean recovery of control samples) / (concentration of cross reactant] × 100

트 Cross-reactivity of ALFIS Vitamin D test with various potential cross-reactants that were tested at specified highest concentration has been shown in the following table.

Potential cross-reactant tested	Concentration of thecross-reactant tested	Percent cross-reactivityobserved
25-hydroxyvitamin D3	50 ng/mL	99.30 – 100.61 %
25-hydroxyvitamin D2	50 ng/mL	98.42 – 98.38 %
3-epi-25-hydroxyvitamin D3	50 ng/mL	20.24 – 22.18 %
24,25-dihydroxyvitamin D3	50 ng/mL	121.59 – 121.91 %
1,25-dihydroxyvitamin D3	100 ng/mL	0.81 – 0.27 %
1,25-dihydroxyvitamin D2	100 ng/mL	0.59 – 0.05 %
Vitamin D3 (Cholecalciferol)	1000 ng/mL	0.1 – 0.04 %
Vitamin D2 (Ergocalciferol)	1000 ng/mL	0.06 – 0.08 %

7) In-house repeatability and reproducibility:

In-house repeatability and reproducibility studies of ALFIS Vitamin D test system । were performed as per CLSI EP05-A3 guideline.
For evaluating in-house repeatability of ALFIS Vitamin D, 3 spiked serum samples । having 25-hydroxyvitamin D concentrations 10, 30, 50 ng/mL were tested with 1 lot of ALFIS Vitamin D Cartridges and 1 ALFIS-3 Analyzer. Each sample was tested in duplicate daily for 20 successive days by the same operator.
For evaluating in-house reproducibility of ALFIS Vitamin D, 3 spiked serum samples । having 25-hydroxyvitamin D concentrations 10, 30, 50 ng/mL were tested with 3

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cartridge lots and ALFIS-3 analyzer/operator pairs. Each sample was tested in quintuplicate daily for 5 successive days.

In these in-house studies, ALFIS Vitamin D test system showed CV < 10% thereby । indicating acceptable repeatability as well as reproducibility.

8) External site-to-site reproducibility:

External site-to-site reproducibility study of ALFIS Vitamin D test system was performed at one in-house research laboratory and two external clinical laboratories in the US as per CLSI EP05-A3 guideline.
3 spiked serum samples having 25-hydroxyvitamin D concentrations 10, 30, 50 ng/mL । were tested with 3 cartridge lots (one lot/site) and 3 ALFIS-3 analyzer-operator (one analyzer-operator per site). Each sample was tested in quintuplicate daily for 5 successive days.
ALFIS Vitamin D test system showed CV < 10% thereby indicating acceptable site-tosite reproducibility.

9) Matrix Comparison:

Matching clinical serum, lithium heparin plasma, and sodium heparin plasm samples obtained from same study subjects were tested with one lot of ALFIS Vitamin D test cartridges on one ALFIS-3 analyzer. Additional 5 spiked samples were tested with the same lot of ALFIS Vitamin D test cartridges and ALFIS-3 analyzer to cover the entire measuring range of ALFIS Vitamin D Test System.
। Regression analysis of the matrix comparison study data showed acceptable commutability of ALFIS Vitamin D Test results across the tested matrices of clinical samples.

Sr.No.	Sample matrices compared	Number ofSamples	Linear regressionequation	Correlationcoefficient
Measuring/reportable range of ALFIS Vitamin D Test = 6 – 100 ng/mL
1	Serum vs. Li-heparin Plasma	45	$y = 0.9797x + 0.3501$	0.9982
2	Serum vs. Na-heparin Plasma	45	$y = 0.98x + 0.3486$	0.9981

9) Reference Interval:

। Reference interval of ALFIS Vitamin D test was evaluated by testing serum samples collected from 299 apparently healthy adults in the United States which comprised 149 and 150 samples meeting specified demographic criteria obtained respectively from two external clinical samples repositories.
The sample sets procured for the reference interval study met the following criteria: ।
- · Age (adults): 21~70 years of age

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· Approximately: 20% central, 20% northern, 60% southern State residents
· Approximately: 50% male, 50% female
At least : 30% collected from African American and 30% from Caucasian

· Approximately: 50% collected during fall, 25% during winter and 25% during summer

· Individuals not taking vitamin D supplements; 30% of individuals did report to taking multivitamins. Note, the vitamin D dosage in multivitamins sold in the US is < 2000 IU Vitamin D)

• Collected after June 2021

Non-parametric reference interval encompassing the central 95% frequency । distribution of study results was determined as per CLSI C28-A3c Standard.
2.5th percentile and 97.5th percentile of distribution of test results was taken as the । lower limit and upper limit respectively of the reference interval.
Adult reference intervals of ALFIS Vitamin D test have been found to be 8.75-43.66 । ng/mL for serum samples.
The manufacturer claims 9.0 43.0 ng/mL as the adult reference interval of ALFIS । Vitamin D test.
However, the user laboratories should establish their own reference intervals for । specific population and/or specific group(s) of population they may cater to.

10) Method Comparison Study:

Method comparison study of ALFIS Vitamin D test was carried out at the in-house laboratory with the predicate device (Elecsys Vitamin D Total III) by testing 120 CDC VDSCP verification samples spanning Total 25-hydroxy Vitamin D concentrations from 6.45 ng/mL to 83.33 ng/mL as confirmed by the Reference Measurement Procedure (RMP) of 25-hydroxyvitamin D i.e., ID-LC-MS/MS at CDC certified laboratory with NIST standard reference material 2972.
The samples were tested with one lot of ALFIS Vitamin D cartridges and one ALFIS i 3 analyzer.
ALFIS Vitamin D test measurements were compared with Total 25-hydroxy Vitamin D concentrations as per the Reference Measurement Procedure (RMP) as well as with the predicate device (Elecsys Vitamin D Total III) test results.
। Weighted Deming regression analysis of the method comparison study data revealed acceptable compatibility of ALFIS Vitamin D test results.

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Sr.No.	Compared test results	Number ofsamplescompared	Linear Regression Equation	CorrelationCoefficient
Measuring/reportable range of ALFIS Vitamin D test = 6.0 - 100.0 ng/mL
1	Reference Measurement Procedure(RMP) vs. ALFIS Vitamin D test(6.45 - 83.33 ng/mL)	120	y = 0.997x + 0.4725(0.9449 - 1.049) (-1.006 - 1.951)	0.978
2	Elecsys Vitamin D Total IIIvs. ALFIS Vitamin D test(6.18 - 79.48 ng/mL)	120	y = 1.006x + 0.05392(0.9756 - 1.036) (-0.7186 - 0.8265)	0.9844

Conclusion:

Based on the intended use, principle and non-clinical as well as clinical performance characteristics described above, ALFIS Vitamin D (with ALFIS-3 analyzer) test system is substantially equivalent to the predicate device Elecsys Vitamin D Total III Assay.

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.