Elecsys Vitamin B12 II assay, Elecsys Vitamin B12 II CalSet

K151786 · Roche Diagnostics · CDD · Sep 24, 2015 · Clinical Chemistry

Device Facts

• Record ID: K151786
• Device Name: Elecsys Vitamin B12 II assay, Elecsys Vitamin B12 II CalSet
• Applicant: Roche Diagnostics
• Product Code: CDD · Clinical Chemistry
• Decision Date: Sep 24, 2015
• Decision: SESE
• Submission Type: Traditional
• Regulation: 21 CFR 862.1810
• Device Class: Class 2

Intended Use

Binding assay for the in vitro quantitative determination of vitamin B12 in human serum and plasma. Measurements obtained by this device are used in the diagnosis and treatment of anemias of gastrointestinal malabsorption. The electrochemiluminescence immunoassay "ECLIA" is intended for use on Elecsys and cobas e immunoassay analyzers. Vitamin B12 II CalSet is used for calibrating the quantitative Elecsys Vitamin B12 II assay on the Elecsys and cobas e immunoassay analyzers.

Device Story

The Elecsys Vitamin B12 II assay is an electrochemiluminescence immunoassay (ECLIA) used on Roche Elecsys and cobas e analyzers to quantify vitamin B12 in human serum or plasma. The device uses recombinant porcine intrinsic factor to bind vitamin B12 in the sample. The assay follows a competitive principle: vitamin B12 in the sample competes with biotinylated vitamin B12 for binding sites on ruthenium-labeled intrinsic factor. The resulting complex binds to streptavidin-coated microparticles. Upon magnetic capture on an electrode, a voltage is applied to induce chemiluminescence, which is measured by a photomultiplier. The output is a quantitative concentration of vitamin B12, used by clinicians to diagnose and manage anemias related to gastrointestinal malabsorption. The assay requires pretreatment of samples to release bound vitamin B12. The system includes a dedicated CalSet for calibration. The device is intended for professional use in clinical laboratory settings.

Clinical Evidence

Bench testing only. Precision evaluated per CLSI EP5-A2 (n=84). Linearity evaluated per CLSI EP6-A (110-2239 pg/mL). Detection limits (LoB 44.3 pg/mL, LoD 89.2 pg/mL, LoQ 129 pg/mL) determined per CLSI EP17-A2. Method comparison with predicate (n=112) yielded y = 0.953x - 11.1 (r=0.942). Interference testing performed per CLSI EP7-A2; hemolysis >100 mg/dL causes falsely depressed results. Reference range established using 120 healthy subjects (median 443 pg/mL).

Technological Characteristics

Electrochemiluminescence immunoassay (ECLIA). Reagents: recombinant porcine intrinsic factor, biotinylated vitamin B12, streptavidin-coated microparticles. Energy source: electrical voltage for chemiluminescence induction. Dimensions/Form: kit-based reagents for automated analyzers. Connectivity: integrated with Elecsys/cobas e immunoassay analyzers. Sterilization: N/A. Software: embedded firmware for instrument control and signal processing.

Indications for Use

Indicated for the in vitro quantitative determination of vitamin B12 in human serum and plasma to aid in the diagnosis and treatment of anemias of gastrointestinal malabsorption.

Regulatory Classification

Identification

A vitamin B12 test system is a device intended to measure vitamin B12 in serum, plasma, and urine. Measurements obtained by this device are used in the diagnosis and treatment of anemias of gastrointestinal malabsorption.

Predicate Devices

• Roche Elecsys Vitamin B12 Immunoassay (k060755)
• Roche Elecsys Vitamin B12 CalSet II (k060755)

Reference Devices

• Elecsys Vitamin B12 assay (k973702)

Submission Summary (Full Text)

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Image /page/0/Picture/1 description: The image shows the logo for the U.S. Department of Health and Human Services. The logo consists of a circular seal with the words "DEPARTMENT OF HEALTH & HUMAN SERVICES • USA" around the perimeter. Inside the circle is a stylized image of three human profiles facing to the right, stacked on top of each other.

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

ROCHE DIAGNOSTICS KELLI TÜRNER REGULATORY AFFAIRS PRINCIPAL 9115 HAGUE ROAD INDIANAPOLIS IN 46250

September 24, 2015

Re: K151786

Trade/Device Name: Elecsys Vitamin B12 II Assay, Elecsys Vitamin B12 II CalSet Regulation Number: 21 CFR 862.1810 Regulation Name: Vitamin B12 test system Regulatory Class: II Product Code: CDD, JIT Dated: June 29, 2015 Received: July 1, 2015

Dear Ms. Turner:

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

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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 [phone] or [phone] or at its Internet address

http://www.fda.gov/MedicalDevices/Resourcesfor You/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 [phone] or [phone] or at its Internet address

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

Sincerely yours.

Katherine Serrano -S

For : Courtney H. Lias 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 : k151786

Device Name Elecsys Vitamin B12 II assay

Indications for Use (Describe)

Binding assay for the in vitro quantitation of vitamin B12 in human serum and plasma. Measurements obtained by this device are used in the diagnosis and treatment of gastrointestinal malabsorption. The electrochemiluminescence immunoassay "ECLIA" is intended for use on Elecsys and cobas e immunoassay analyzers.

| Type of Use (Select one or both, as applicable) | |
|-------------------------------------------------------------------------------|-----------------------------------------------------------------------------|
| <div> <span> Prescription Use (Part 21 CFR 801 Subpart D) </span> </div> | <div> <span> Over-The-Counter Use (21 CFR 801 Subpart C) </span> </div> |

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# Indications for Use

510(k) Number:k151786

Device Name Elecsys Vitamin B12 II CalSet

Indications for Use (Describe)

Vitamin B12 II CalSet is used for calibrating the quantitative Elecsys Vitamin B12 II assay on the Elecsys and cobas e immunoassay analyzers.

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)

### 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 [e-mail]

"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."

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# Elecsys Vitamin B12 II Test System 510(k) Summary:k151786

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

| Submitter Name | Roche Diagnostics |
|----------------------------|-------------------------------------------------------------------------------------------------------------------------------------------------------------------------|
| Address | 9115 Hague Road<br>Indianapolis, IN 46250 |
| Contact | Kelli Turner<br>Phone: [phone]<br>FAX: [phone]<br>Email: [e-mail] |
| Date Prepared | 06/29/2015 |
| Proprietary Name | 1. Elecsys Vitamin B12 II assay,<br>2. Elecsys Vitamin B12 II CalSet |
| Common Name | 1. Vitamin B12 II assay<br>2. Vitamin B12 II CalSet |
| Classification Name | 1. Radioassay, Vitamin B12<br>2. Secondary, calibrator |
| Product Codes | 1. CDD, 862.1810<br>2. JIT, 862.1150 |
| Predicate Devices | Elecsys Vitamin B12 Assay (k060755) |
| Establishment Registration | Roche Diagnostics GmbH in Mannheim, Germany, is 9610126<br>Roche Diagnostics GmbH in Penzberg, Germany, is 9610529<br>Roche Diagnostics in the United States is 1823260 |

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#### 1. DEVICE DESCRIPTION

The Elecsys Vitamin B12 II assay employs a competitive test principle using intrinsic factor specific for vitamin B12. Vitamin B12 in the sample competes with the added vitamin B12 labeled with biotin for the binding sites on the ruthenium-labeled intrinsic factor complex.

Results are determined via a calibration curve which is instrument specifically generated by 2-point calibration and a master curve provided via reagent barcode.

#### 1.1. Reagents

The reagent working solutions include:

- the rackpack (kit placed on instrument) .
- Streptavidin coated microparticles, .
- Reagent 1 (ruthenium labeled intrinsic factor) and .
- Reagent 2 (vitamin B12 labeled biotin). .
- Pretreatment 1 (Dithiothreitol) .
- Pretreatment 2 (sodium hydroxide, sodium cyanide) .

#### Calibrator 1.2.

The Vitamin B12 II CalSet is a lyophilized human serum matrix with added vitamin B12 in two concentration ranges.

The CalSet includes:

- Cal 1 (approximately 250 pg/mL vitamin B12 in a Human serum matrix) .
- Cal 2 (approximately 1500 pg/mL vitamin B12 in a Human serum matrix) .

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#### 2. INDICATIONS FOR USE

## Elecsys B12 II assay

Binding assay for the in vitro quantitative determination of vitamin B12 in human serum and plasma. Measurements obtained by this device are used in the diagnosis and treatment of anemias of gastrointestinal malabsorption.

The electrochemiluminescence immunoassay "ECLIA" is intended for use on Elecsys and cobas e immunoassay analyzers.

# Elecsys B12 II CalSet

Vitamin B12 II CalSet is used for calibrating the quantitative Elecsys Vitamin B12 II assay on the Elecsys and cobas e immunoassay analyzers.

#### TECHNOLOGICAL CHARACTERISTICS 3.

Table 1: Assay Comparison: General Assay Features

| Feature | Predicate Device: Elecsys Vitamin B12<br>(k060755) | Candidate Device: Elecsys Vitamin B12 II Assay |
|-----------------------------------------|-------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------|------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------|
| Intended Use/<br>Indications for<br>Use | Binding assay for the in vitro quantitative<br>determination of vitamin B12 in human serum<br>and plasma.<br>The electrochemiluminescence immunoassay<br>"ECLIA" is intended for use on Elecsys and<br>cobas e immunoassay analyzers.<br>A Vitamin B12 test system is a device intended<br>to measure Vitamin B12 in serum, plasma.<br>Measurements obtained by this device are<br>used in the diagnosis and treatment of anemias<br>of gastrointestinal malabsorption. | Binding assay for the in vitro quantitative<br>determination of vitamin B12 in human serum<br>and plasma. Measurements obtained by this<br>device are used in the diagnosis and treatment<br>of anemias of gastrointestinal malabsorption.<br>The electrochemiluminescence immunoassay<br>"ECLIA" is intended for use on Elecsys and<br>cobas e immunoassay analyzers. |
| Assay Protocol | The Elecsys Vitamin B12 assay employs a<br>competitive test principle using<br>intrinsic factor specific for vitamin B12. Vitamin<br>B12 in the sample competes with the added<br>vitamin B12 labeled with biotin for the binding<br>sites on the ruthenium- labeled intrinsic factor<br>complex. | Same. |
| Detection<br>Protocol | Electrochemiluminescent Assay | Same. |
| Applications | 27-minute application | Same. |
| Instrument<br>Platform | Elecsys and cobas e immunoassay analyzers. | Same. (submission for cobas e 411) |
| Sample Volume | 15 µL | Same. |
| Sample<br>Type | Human serum and plasma. | Same. |
| Reagents | Competition principle. Total duration of assay:<br>27 minutes | Same. |
| Calibrator | Elecsys Vitamin B12 CalSet II | Elecsys Vitamin B12 II CalSet |

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| Feature | Predicate Device: Elecsys Vitamin B12<br>(k060755) | Candidate Device: Elecsys Vitamin B12 II<br>Assay |
|-----------------------------------|----------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------|----------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------|
| Calibration<br>Interval | Calibration must be performed once per<br>reagent lot using fresh reagent (i.e. not more<br>than 24 hours since the reagent kit was<br>registered on the analyzer). Renewed<br>calibration is recommended as follows:<br>• After 1 month (28 days) when using the<br>same reagent lot.<br>• After 7 days (when using the same<br>reagent kit on the analyzer).<br>• As required: e.g. quality control findings<br>outside the defined limits | Same. |
| Controls | Elecsys PreciControl Varia | Same |
| Traceability /<br>Standardization | Standardized against the Elecsys Vitamin B12<br>assay (k973702) | Standardized against the Elecsys Vitamin B12<br>assay (k060755).<br>Accuracy to WHO Standard 03/178 |
| Reagent<br>Stability | Unopened: 2-8°C - Up to the stated expiration<br>date.<br>After Opening at 2-8°C - 12 weeks<br>On the Analyzers – 5 weeks | Unopened: 2-8°C - Up to the stated expiration<br>date.<br>After Opening at 2-8°C - 12 weeks<br>On the Analyzers - 5 weeks onboard or 60<br>days when stored alternatively in the<br>refrigerator and on the analyzer, with the total<br>time onboard on the analyzer not exceeding 10<br>x 8 hours |
| Measuring<br>Range | 30.0 - 2000 pg/mL | 150- 2000 pg/mL |

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| Feature | Predicate Device: Elecsys Vitamin B12<br>(k060755) | | | Candidate Device: Elecsys Vitamin B12 II Assay | | | | |
|---------------------------|----------------------------------------------------|------------------------------------|----------------------------------------------|-----------------------------------------------------------------------------------------------------------------------|----------------|------------------------------------|----------------------------------------------|------|
| Precision | cobas e 411:<br>Within-run | | | cobas e 411:<br>Within-run (will be labeled Repeatability) | | | | |
| | Sample | Mean (pg/mL) | SD | CV | Sample | Mean (pg/mL) | SD | CV |
| | HS 1 | 192 | 11.3 | 5.9% | HS 1 | 176 | 8.86 | 5.0% |
| | HS 2 | 358 | 20.1 | 5.6% | HS 2 | 405 | 13 | 3.2% |
| | HS 3 | 864 | 29.1 | 3.3% | HS 3 | 960 | 19.7 | 2.1% |
| | HS 4 | 1625 | 62.1 | 3.8% | HS 4 | 1230 | 27.4 | 2.2% |
| | PCV0 | 221 | 10.2 | 4.6% | HS 5 | 1940 | 40.9 | 2.1% |
| | PCV1 | 467 | 18.5 | 4.0% | PCV0 | 229 | 8.96 | 3.9% |
| | PCV2 | 958 | 31.1 | 3.3% | PCV1 | 447 | 12.2 | 2.7% |
| | | | | | PCV2 | 934 | 20.2 | 2.2% |
| | Total | | | Total (will be labeled Intermediate precision) | | | | |
| | Sample | Mean (pg/mL) | SD | CV | Sample | Mean (pg/mL) | SD | CV |
| | HS 1 | 192 | 19.8 | 10.3% | HS 1 | 176 | 12.7 | 7.2% |
| | HS 2 | 358 | 27.7 | 7.7% | HS 2 | 405 | 17.5 | 4.3% |
| | HS 3 | 864 | 40.1 | 4.5% | HS 3 | 960 | 31.0 | 3.2% |
| | HS 4 | 1625 | 65.1 | 4.0% | HS 4 | 1230 | 46.4 | 3.8% |
| | PCV0 | 221 | 10.2 | 4.6% | HS 5 | 1940 | 72.6 | 3.7% |
| | PCV1 | 467 | 18.5 | 4.0% | PCV0 | 229 | 12.4 | 5.4% |
| | PCV2 | 958 | 31.1 | 3.3% | PCV1 | 447 | 18.6 | 4.2% |
| | | | | | PCV2 | 934 | 38.4 | 4.1% |
| Analytical<br>Sensitivity | Lower detection limit = 30.0 pg/mL | | | Limit of Blank (LoB): = 50 pg/mL Limit<br>of Detection (LoD): = 100 ng/mL<br>Limit of Quantitation (LoQ): = 150 pg/mL | | | | |
| Analytical<br>Specificity | Cross reactant | concentration<br>tested<br>(ng/mL) | Highest cross-<br>reactivity<br>observed (%) | | Cross reactant | concentration<br>tested<br>(ng/mL) | Highest cross-<br>reactivity<br>observed (%) | |
| | Cobinamide | 200 | 0.024 | | Cobinamide | 210 | 0.003 | |
| Linearity | 30 to 2000 pg/mL | | | 150 to 2000 ng/mL | | | | |

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| Feature | Predicate Device: Elecsys Vitamin B12<br>(k060755) | Candidate Device: Elecsys Vitamin B12 II<br>Assay |
|-------------|----------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------|----------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------|
| Limitations | The assay is unaffected by:<br>• Bilirubin < 65 mg/dL<br>• Hemolysis < 1.0 g/dL<br>• Lipemia < 1500 mg/dL<br>• Biotin < 50 ng/mL<br>• Rheumatoid factors < 1500 IU/mL<br>• In vitro tests were performed on<br>54 commonly used pharmaceuticals. No<br>interference with the assay was found.<br>• In rare cases, interference due to<br>extremely high titers of antibodies to<br>analyte-specific antibodies, streptavidin<br>or ruthenium can occur. These effects<br>are minimized by suitable test design.<br>For diagnostic purposes, the results should<br>always be assessed in conjunction with RBC<br>folate, the patient's medical history, clinical<br>examination and other findings. | The assay is unaffected by:<br>• Bilirubin ≤ 65 mg/dL<br>• Hemolysis ≤ 0.1 g/dL<br>• Lipemia ≤ 1500 mg/dL<br>• Biotin ≤ 50 ng/mL<br>• Rheumatoid factors < 1500 IU/mL<br>• IgG ≤ 28 g/dL<br>• IgM ≤ 10 g/dL<br>• IgA ≤ 16 g/dL<br>• In vitro tests were performed on<br>16 commonly used pharmaceuticals. No<br>interference with the assay was found.<br>• In rare cases, interference due to<br>extremely high titers of antibodies to<br>analyte-specific antibodies, streptavidin<br>or ruthenium can occur. These effects<br>are minimized by suitable test design.<br>• Because intrinsic factor is typically used<br>as the binding protein in serum vitamin<br>B12 assays, anti-intrinsic factor<br>antibodies (which are common in<br>pernicious anemias) can lead to<br>elevated vitamin B12 measurement<br>values.<br>• The Elecsys Vitamin B12 II assay is<br>designed to avoid interference due to<br>anti-intrinsic factor antibodies.<br>For diagnostic purposes, the results should<br>always be assessed in conjunction with the<br>patient's medical history, clinical examination<br>and other findings.<br>Note: the presence of immunoglobulin-vitamin<br>B12 complexes may cause unexpectedly high<br>values of vitamin B12. |

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| Feature | Predicate Device: Elecsys Vitamin B12<br>(k060755) | | | | Candidate Device: Elecsys Vitamin B12 II Assay | | | | | | | |
|--------------------------|----------------------------------------------------|-----|--------|----------------|------------------------------------------------|---------|----------------------------------|-------------------|--------|--------|-------------------------|---------|
| Reference<br>range study | Country | (N) | Median | | 2.5th-97.5th percentile | | Subject cohort | (N) | Median | | 2.5th-97.5th percentile | |
| | USA | 178 | pg/mL | Pmol/L | pg/mL | pmol/L | Apparently healthy male & female | 120 | pg/mL | pmol/L | pg/mL | pmol/L |
| | | | 463 | 342 | 211-946 | 156-698 | | | 443 | 327 | 232-1245 | 171-919 |
| Method<br>Comparison | n = 120 | | | Passing/Bablok | | | | Linear regression | | | | |
| | Min = 156 pg/mL | | | | | | | | | | | |
| | Max = 1753 pg/mL | | | | | | | | | | | |
| | Slope | | | 0.950 | | | | 0.984 | | | | |
| | Intercept | | | -9.83 | | | | -26.7 | | | | |
| | Tau/r | | | 0.941 | | | | 0.996 | | | | |
| | Bias at 200 pg/mL | | | -9.88 | | | | -15.0 | | | | |

#### Table 2: Assay Comparison: Labeled Performance Characteristics

HS= Human Serum

PCV0=PreciControl Varia level 0

PCV1=PreciControl Varia level 1

PCV2=PreciControl Varia level 2

#### Table 3: CalSet Comparison

| Characteristic | Predicate device: Elecsys Vitamin B12<br>CalSet II | Candidate device: Elecsys Vitamin B12 II |
|----------------|--------------------------------------------------------------------------------------------------------------------------------------------------|--------------------------------------------------------------------------------------------------------------------------------------------------------|
| Intended Use | Vitamin B12 CalSet II is used for calibrating the<br>quantitative Elecsys Vitamin B12 assay on the<br>Elecsys and cobas e immunoassay analyzers. | Vitamin B12 II CalSet is used for calibrating the<br>quantitative Elecsys Vitamin B12 II assay on<br>the Elecsys and cobas e immunoassay<br>analyzers. |
| Analyte | Vitamin B12 | Same |
| Matrix | Human serum matrix | Same |
| Levels | Two | Same |
| Target Ranges | Cal 1: 250 pg/mL<br>Cal 2: 1500 pg/mL | Same. |
| Format | Lyophilized | Same |

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| Characteristic | Predicate device: Elecsys Vitamin B12<br>CalSet II | Candidate device: Elecsys Vitamin B12 II |
|----------------|----------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------|------------------------------------------|
| Handling | Carefully dissolve the contents of one bottle by<br>adding exactly 1.0 mL of distilled or deionized<br>water and allow to stand closed for 15 minutes<br>to reconstitute. Mix carefully, avoiding foam<br>formation.<br>Transfer aliquots of the reconstituted calibrators<br>into empty labeled snap- cap bottles<br>(CalSet Vials). Attach the supplied labels to the<br>additional bottles. Store the aliquots<br>immediately at - 20 °C.<br>Perform only one calibration procedure<br>per aliquot. | Same. |

#### 4. NON-CLINICAL PERFORMANCE EVALUATION

Non-clinical performance evaluations for the Elecsys Vitamin B12 II executed with the study briefly summarized.

#### 4.1. Precision

The precision results were obtained using serum samples evaluated on the cobas e 411 Immunoassay Analyzer. Within-run precision (repeatability) and total imprecision (intermediate precision) were determined according to CLSI Guideline EP5-A2. The protocol included testing 2 replicates per run, 2 runs per day for 21 days.

Specifications:

| | Repeatability | Intermediate precision |
|-------------|---------------|------------------------|
| ≤ 200 pg/mL | SD ≤ 14 pg/mL | SD ≤ 24 pg/mL |
| > 200 pg/mL | CV ≤ 7 % | CV ≤ 12 % |

All results met the pre-defined acceptance criteria for repeatability and intermediate precision.

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#### 4.2. Limit of Blank

For the analytical sensitivity studies, 2 cobas e 411 analyzers and 3 lots of reagents were used. The Limit of Blank (LoB) was determined using a buffered human serum albumin matrix similar to human serum (no analyte added). A total of n = 60 LoB measurements were made (5 replicates, 2 runs per day on 2 instruments over 3 days). The LoB was calculated according to CLSI EP17-A2 (non-parametric approach). Acceptance criterion: LoB ≤ 50 pg/mL

#### Limit of Detection 4.3.

For the analytical sensitivity studies, 2 cobas e 411 analyzer and 3 lots of reagents were used. The Limit of Detection (LoD) was determined using 5 low-level human serum samples (native and diluted). A total of n = 60 LoD measurements were made (5 samples, 2 runs per day on 2 instruments over 3 days). The LoD was calculated according to CLSI EP17-A2 (chapter 5.3.3.2). Acceptance criterion: LoD ≤ 100 pg/mL

#### Limit of Quantitation 4.4.

The Limit of Quantitation (LoQ) was determined using a minimum set of seven human serum samples, three reagent lots on one cobas e 411 analyzer. The Limit of Quantitation (LoQ) was determined in accordance with CLSI Guideline EP17-A2. Each sample was analyzed in replicates of 5, one run per day over 5 days. Acceptance criterion: LoQ (Imprecision) ≤ 20 % at 150 pg/mL.

#### 4.5. Dilution

To demonstrate the Vitamin B12 II assay dilution study, four different dilutions with dilution factors between 1:1.5 and 1:3 were prepared.

Five serum samples were spiked with vitamin B12 to concentrations exceeding the measuring range. The samples were diluted with Elecsys Diluent Universal and recovery was investigated on the cobas e 411. The vitamin B12 concentrations of the undiluted samples were calculated by multiplying the result of the diluted samples using the appropriate dilution factor.

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#### 4.6. Linearity

The linearity results were obtained with serum samples on the cobas e 411 Immunoassay analyzer. Linearity was determined according to CLSI Guideline EP6-A. All results met the predefined acceptance criteria for linearity. The linearity results support a claimed measuring range.

Acceptance criteria:

- Significant level for deviation to higher order polynomial: 5% .
- Limits for deviation to higher order polynomial regression .
- ≤ 200 pg/mL: ± 20 pg/mL .
- > 200 pg/mL: ± 10 % .

#### 4.7. Analytical Specificity

The specificity was determined using two human serum samples (single donors, native) spiked with potential cross-reactant compounds. The analyte concentration of the samples was at 129 and 550 pg/mL Vitamin B12. Four levels of the cross-reactant were prepared and measured in duplicate. The spiked and non-spiked samples were tested in duplicates on the cobas e 411 Immunoassay Analyzer. Cross-reactivity results will be reported in the method sheet.

#### Endogenous Interferences 4.8.

The effect on quantitation of analyte in the presence of endogenous interfering substances was determined on the cobas e 411 Immunoassay analyzer using human serum samples (single donors, native as well as spiked). For each interfering substance (Biotin, Lipemia, Hemoglobin, Bilirubin, Rheumatoid Factor, IgG/IgM/IgA) three human serum samples containing low, mid and high concentrations of Vitamin B12 were tested. The recovery for each sample was calculated by comparison to the reference sample. Predefined acceptance criterion was met. The claims included in the method sheet were set set to the concentration without observed interference.

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Acceptance criterion:

- . < 200 pg/mL: ± 20 pg/mL of unspiked reference value
- > 200 pg/mL: ± 10% of unspiked reference value .

#### Exogenous Interferences- Drugs 4.9.

The effect on quantitation of analyte in the presence of drugs was determined by comparing values obtained from samples spiked with 16 pharmaceutical compounds spiked into two human serum samples (single donors, native) and tested on the cobas e 411 Immunoassay Analyzer. The analyte concentration of the samples were approximately 200 and 1200 pg/mL. The drug concentrations tested are in accordance with the recommendation (if available) given in the CLSI guideline EP7-A2. When concentrations are not given in the guideline, concentrations of at least 3-times of the maximum recommended daily dose were tested. The two serum samples were divided into aliquots and spiked with the potential interferents. The reference sample without interferent was spiked with the respective amount of solvent only.

The concentration of the spiked aliquots was determined in 8-fold determination and compared to the Vitamin B12 concentration determined for the reference aliquot (also in 8-fold determination). Acceptance criterion: ± 10% of the reference value (unspiked sample).

# 4.10. Exogenous Interferences- Anticoagulants

The effect on quantitation of analyte in the presence of anticoagulants with the Elecsys Vitamin B12 II Immunoassay was determined by comparing values obtained from samples (single donors, native as well as spiked) drawn into Serum, Li-Heparin, Na-Heparin, K2-EDTA-, K3-EDTA-plasma primary tubes and Li-Heparin Plasma Gel Separation Tubes. A minimum of 90 serum/plasma pairs per sample material were tested in singleton with one reagent lot on one cobas e 411 Immunoassay Analyzer. Potential effects are assessed by regression analysis.

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Acceptance criterion:

- . Slope (Passing/Bablok): 0.9 – 1.1
- Intercept (Passing/Bablok): <±100 pg/mL .
- coefficient of correlation Pearson's r (linear regression): ≥ 0.95 .
- Bias at 200 pg/mL: ± 10 % .

# 4.11. Method Comparison

A method comparison was performed using the current Elecsys Vitamin B12 assay (cleared under K060755) as predicate device.

A total of 120 human serum samples (all single donors, native as well as spiked) were measured in singleton covering the entire measuring range.

The study was performed on the cobas e 411 analyzer over 3 runs using the current Elecsys Vitamin B12 assay (X) and the updated Elecsys Vitamin B12 II assay (Y). Regression analysis was performed.

Acceptance criteria:

- 1.00 ± 0.05 . Slope (Passing/Bablok):
- Correlation (linear regression) Pearson's r: . > 0.95
- Intercept (Passing/Bablok): < ± 30 pg/mL .
- Bias at 200 pg/mL: ≤± 10 % .

# 4.12. Reagent Stability

To test reagent stability, four studies were executed with three studies completed.

# 4.12.1. Study 1. Reagent stability refrigerator/onboard (60 days)

Reagent stability refrigerator / onboard was tested on one cobas e 411 Immunoassay Analyzer.

A fresh reagent rackpack (kit) was placed on the analyzer and calibrated. Reference values for the five human serum (HS) samples and three controls were determined in duplicate. After

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measurement the rackpack was removed from the analyzer and kept at 2-8 °C for 64 days. During this period of time, the kit was stressed alternately at 20°C + 3°C (on-board condition) 10 times for 8 hours (80 hours in total). On day 8, 15, 29, 50 and day 64, the same samples were measured with this reagent kit kept under alternating storage conditions (refrigerator /on-board) using the calibration curves established on day 0, 8, 22, 43 and 57, respectively. Acceptance criterion for recovery was compared to day 0 value.

# 4.12.2. Study 2. Reagent stability after first opening at 2-8°C (84 days)

Reagent stability after first opening for the Elecsys Vitamin B12 II assay was tested on one cobas e 411 Immunoassay Analyzer.

A fresh reagent rackpack was placed on the analyzer and calibrated. Reference values for the five human serum (HS) samples and three controls were determined in duplicate. After measurement the kit was removed from the analyzer and kept at 2-8 °C up to 92 days. After 36, 64 and 92 days the kit was placed on the analyzer again, calibrated and the test samples were determined. Acceptance criterion for recovery was compared to day 0 value.

# 4.12.3. Study 3. On board reagent stability (35 days)

Reagent On-board Stability and Calibration Stability for the Elecsys Vitamin B12 II assay was tested on one cobas e 411 Immunoassay Analyzer. A fresh Reagent Rack-Pack was placed on the analyzer and calibrated. Reference values for the five human serum (HS) samples and three controls were measured in duplicate on day 0. On day 8, 22 and day 36, the same samples were measured with the same reagent kit kept at 20°C + 3°C (on-board condition) using the calibration curves established on day 0, 15 and 29, respectively. Acceptance criterion for recovery was compared to day 0 value.

# 4.12.4. Study 4: A real-time stability study is ongoing to support shelf-life stability claim.

In the ongoing real-time stability study, the Elecsys Vitamin B12 II reagent is stored at 2-8°C. The stored assay reagents are tested at time point T=0 (after manufacturing) and at specified intervals over the shelf life of the device up to the planned shelf life plus one month. Testing will be performed using PreciControl Varia 3. Level 0. 1 and 2 (lyopylized, stored at -20℃) and in addition three human serum samples (stored at -80°C).

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Data for the time-points at 0, 7, 10, 13, 19 and 25 months tested in duplicate will be available. The average on-test recovery value will be calculated as percent recovery compared to the reference value:

- assigned value for PreciControl Varia 3, Level 0, 1 and 2 .
- value measured at T=0 for the human serum samples .

The acceptance criterion is recovery of 80 -120 % for PC Varia and the human serum samples.

# 4.13. Sample Stability

To test sample stability, three studies were completed. Because these studies are not analyzer dependent, and the assay is a global product currently available rest of world (ROW), the studies were executed on the cobas e 601. Study results can be applied to the cobas e 411 since sample stability is independent of the analyzer. For each study, the samples used were all single donors (native, spiked and diluted) and the following acceptance criteria was applied.

Acceptance criteria for recovery compared to the reference value:

- ≤ 200 pg/mL: ± 28 pg/mL .
- > 200 pg/mL: ± 14 % .

# 4.13.1. Study 1. Sample stability at 2-8°C

Five samples for each sample type (Serum, K2-EDTA-, K3-EDTA-, Li-Heparin- and Na-Heparin plasma) were aliquoted and measured after storage at 2-8℃ for 49 hours. Measurements were performed with three-fold determination and recovery was calculated as percent of the reference value.

# 4.13.2. Study 2. Sample stability at Room Temperature (15-25°C)

Five samples for each sample type (Serum. K2-EDTA-, K3-EDTA-, Li-Heparin- and Na-Heparin plasma) were aliquoted and measured after storage at 15-25°C for 3 hours. The aliguot for the reference value was stored at -80°C and measured at the same time point.

Measurements were performed with three-fold determination and recovery was calculated as percent of the reference value.

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# 4.13.3. Study 3. Sample stability -15 to -25°C

Five samples for each sample type (Serum, K2-EDTA-, K3-EDTA-, Li-Heparin- and Na-Heparin plasma) were aliquoted and measured after storage at -15 to -25℃ for 57 days. The aliquot for the reference value was stored at -80°C and measured at the same time point. Measurements were performed with three-fold determination and recovery was calculated as percent of the reference value.

# 4.14. Calibrator studies

The Elecsys Vitamin B12 II CalSet was evaluated for value assignment, reconstitution and stability.

# 4.14.1. Value assignment

Value assignment testing was conducted and passed pre-defined acceptance criteria. The target values for the two levels of the Vitamin B12 II CalSet kit are chosen to obtain the best fit with the Master Calibration Curve, together with the Rodbard curve parameters encoded in the reagent barcode. For each Elecsys Vitamin B12 II CalSet lot manufactured, the calibrators are run in duplicate on at least three (3) cobas e 411 analyzers and at least three (3) cobas e 601/MODULAR ANALYTICS E170 analyzers with all Vitamin B12 II reagent lots available. The assigned value of each calibrator is defined as the mean value obtained over at least six (6) runs on at least three (3) analyzers) of the respective calibrator.

Measurement values for PreciControl Varia (Levels 0, 1 & 2), a multi-analyte control recommended for use to monitor accuracy and precision of specified analytes, are read from the calibration curves generated. The pre-defined acceptance criteria for PreciControl Varia have to be met to release the Assigned Values for Vitamin B12 II CalSet.

# 4.14.2. Reconstitution

Reconstitution time for the lyophilized Vitamin B12 II CalSet was tested. Two sets of Vitamin B12 II CalSet were reconstituted, one for 15 minutes and the other for 30 minutes. Signal recovery after 30 minutes reconstitution was compared to the signal value after 15 minutes.

Vitamin B12 II CalSet was evaluated in duplicate on the cobas e 411 analyzer as a reference.

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The acceptance criterion was 90 to 110 % signal recovery of the reference material value.

# 4.14.3. Stability

Three studies were performed in order to verify the stability claims for the Vitamin B12 II CalSet. Stability studies after reconstitution and an accelerated stability study were completed on the cobas e 411.

# 4.14.3.1. Study 1- Stability at -20ºC (after reconstitution)

The on-test and reference materials were tested in duplicate on the cobas e 411. The on-test material was reconstituted and stored in closed vials for 85 days at -20°C. The on-test signal recovery was calculated as percent of the reference value.

The acceptance criterion was 90 to 110 % signal recovery of the reference material value.

# 4.14.3.2. Study 2- Stability at 2 to 8°C after reconstitution

The on-test and reference materials were tested in duplicate on the cobas e 411. The on-test material was reconstituted and stored in closed vials for 73 hours at 2-8°C. The on-test signal recovery was calculated as percent of the reference value.

The acceptance criterion was 90 to 110 % signal recovery of the reference material value.

# 4.14.3.3. Study 3- Onboard stability at 20-25℃ after reconstitution

The on-test and reference materials were tested in duplicate on the cobas e 411. The on-test material was reconstituted and stored in open vials for 6 hours at 20-25°C. The on-test signal recovery was calculated as percent of the reference value.

The acceptance criterion was 90 to 110 % signal recovery of the reference material value.

# 4.14.3.4. Real-time Stability

Since there was no change in formulation and target values to the Vitamin B12 II CalSet the realtime stability data (30 months at 2-8°C) of the current Vitamin B12 CalSet II is applicable.

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#### CLINICAL PERFORMANCE EVALUATION 5.

Clinical samples were collected at four sites in the United States in order to establish the reference range values for the Elecsys Vitamin B12 II assay. Reference ranges for apparently healthy males and apparently healthy females were determined using the median value and the 2.5th - 97.5th percentiles (pg/mL) as lower and upper limit of normal, respectively. The evaluation was done at one site in Germany (Roche Diagnostics GmbH, R&D department, Penzberg) with one reagent lot (MP) using one cobas e 411 analyzer. Samples were all native human serum samples measured in singleton, over 2 runs for 2 days.

#### 6. CONCLUSIONS

The information provided in this Premarket Notification [510(k)] will support a determination of substantial equivalence for the Elecsys Vitamin B12 II Test System for the measurement of vitamin B12 in serum and plasma samples The data supports a safe, effective device which performs as well as or better than the predicate device.