AXSYM HOLO TC REAGENT KIT, MODEL 3L83-20; STANDARD CALIBRATOR KIT, MODEL 3L83-01; CONTROL KIT, MODEL 3L83-10

K062467 · Axis-Shield Diagnostics, Ltd. · CDD · Nov 22, 2006 · Clinical Chemistry

Device Facts

• Record ID: K062467
• Device Name: AXSYM HOLO TC REAGENT KIT, MODEL 3L83-20; STANDARD CALIBRATOR KIT, MODEL 3L83-01; CONTROL KIT, MODEL 3L83-10
• Applicant: Axis-Shield Diagnostics, Ltd.
• Product Code: CDD · Clinical Chemistry
• Decision Date: Nov 22, 2006
• Decision: SESE
• Submission Type: Traditional
• Regulation: 21 CFR 862.1810
• Device Class: Class 2

Indications for Use

AxSYM HoloTC assay is a Microparticle Enzyme Immunoassay (MEIA) for the quantitative determination of holotranscobalamin (vitamin B12 bound to transcobalamin) in human serum and plasma on the AxSYM System. HoloTC is used as an aid in the diagnosis and treatment of vitamin B₁₂ deficiency. The AxSYM HoloTC Standard Calibrators are for the standard calibration of the AxSYM System when used for the quantitative determination of holotranscobalamin (HoloTC) in human serum and plasma. The AxSYM HoloTC Controls are for the use in quality control to monitor the accuracy and precision of the HoloTC assay when used for the quantitative determination of holotranscobalamin (HoloTC) in human serum and plasma on the AxSYM System. For in vitro diagnostic use.

Device Story

AxSYM HoloTC is a Microparticle Enzyme Immunoassay (MEIA) performed on the AxSYM System. Input: human serum or plasma samples. Process: sample and anti-HoloTC monoclonal antibody-coated microparticles form antigen-antibody complexes; complexes bind to a glass fiber matrix; alkaline phosphatase-labeled conjugate is added; substrate (4-Methylumbelliferyl Phosphate) is introduced; alkaline phosphatase catalyzes substrate to fluorescent 4-Methylumbelliferone. Output: fluorescent signal measured by MEIA optical assembly, proportional to HoloTC concentration. Used in clinical laboratory settings by trained personnel. Results aid clinicians in diagnosing and managing vitamin B12 deficiency.

Clinical Evidence

No clinical diagnostic studies performed. Evidence consists of analytical performance validation: precision (total CV 6.3-8.5%), linearity (R² 0.97-0.99), analytical sensitivity (≤1 pmol/L), and method comparison against predicate RIA (n=204, slope 1.02, r=0.90). Reference interval established using 281 healthy donors (19.1-119.3 pmol/L).

Technological Characteristics

Microparticle Enzyme Immunoassay (MEIA) technology. Uses anti-HoloTC monoclonal antibody-coated microparticles, alkaline phosphatase-labeled conjugate, and 4-Methylumbelliferyl Phosphate substrate. Measurement via MEIA optical assembly (fluorescence). System-based automated immunoassay.

Indications for Use

Indicated for the quantitative determination of holotranscobalamin (HoloTC) in human serum and plasma to aid in the diagnosis and treatment of vitamin B12 deficiency.

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

• Axis-Shield HoloTC Radioimmunoassay (K030655)

Submission Summary (Full Text)

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# 510(k) SUBSTANTIAL EQUIVALENCE DETERMINATION DECISION SUMMARY ASSAY ONLY TEMPLATE

A. 510(k) Number:
k062467

B. Purpose for Submission:
New device

C. Measurand:
Vitamin B₁₂

D. Type of Test:
Quantitative microparticle enzyme immunoassay (MEIA)

E. Applicant:
Aixis-Shield Diagnostics, Ltd.

F. Proprietary and Established Names:
Axis-Shield HoloTC assay

G. Regulatory Information:
| Product Code | Classification | Regulation Section | Panel |
| --- | --- | --- | --- |
| CDD | II | §862.1810 Vitamin B₁₂ test system | Chemistry |
| JIT | II | §862.1150 Calibrator secondary | Chemistry |
| JJX | I | §862.1660 Single (specified) analyte controls (assayed and unassayed) | Chemistry |

H. Intended Use:
1. Intended use(s):
See indications for use below.

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2. Indication(s) for use:

Indications for Use:

Reagents

AxSYM HoloTC assay is a Microparticle Enzyme Immunoassay (MEIA) for the quantitative determination of holotranscobalamin (vitamin B12 bound to transcobalamin) in human serum and plasma on the AxSYM System. HoloTC is used as an aid in the diagnosis and treatment of vitamin B₁₂ deficiency.

Calibrators

The AxSYM HoloTC Standard Calibrators are for the standard calibration of the AxSYM System when used for the quantitative determination of holotranscobalamin (HoloTC) in human serum and plasma.

Controls

The AxSYM HoloTC Controls are for the use in quality control to monitor the accuracy and precision of the HoloTC assay when used for the quantitative determination of holotranscobalamin (HoloTC) in human serum and plasma on the AxSYM System.

For in vitro diagnostic use.

3. Special conditions for use statement(s):

For prescription use only.

4. Special instrument requirements:

Abbott AxSYM®

I. Device Description:

The AxSYM® HoloTC Reagents, Calibrators and Controls are designed to be used in the AxSYM® HoloTC assay on the AxSYM® system.

Reagents:

The AxSYM® HoloTC assay is a Microparticle Enzyme Immunoassay (MEIA) for the quantitative determination of HoloTC in human plasma on the AxSYM System.

Each HoloTC Reagent kit contains 1 bottle of each component of Wash Buffer, Conjugate, Microparticles and Matrix Cell Wash.

- Microparticles – 1 Bottle (8.4 mL) Anti-HoloTC (Mouse, Monoclonal) Antibody Coated Microparticles in TRIS buffer with protein (Bovine) stabilizers.

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Minimum concentration: 0.025% solids (w/v). Preservative: Sodium Azide. (Reagent Bottle 1).

- Conjugate - 1 Bottle (13.3 mL) Anti-TC Antibody: Alkaline Phosphatase Conjugate in TRIS buffer with protein (Bovine) stabilizers. Minimum concentration: 0.1 µg/mL. Preservative: Sodium Azide. (Reagent Bottle 2).
- Wash Buffer - 1 Bottle (14.2 mL) Wash Buffer containing detergent. Preservative: ProClin 300. (Reagent Bottle 3).
- Matrix Cell Wash - 1 Bottle (33.0mL) Matrix Cell Wash. Preservatives: Sodium Azide and Antimicrobial Agents. (Reagent Bottle 4).

## Calibrators:

The AxSYM® HoloTC Standard Calibrators are for the standard calibration of the AxSYM System when used for the quantitative determination of HoloTC in human serum and plasma.

Each HoloTC Standard Calibrator kit contains 6 bottles of AxSYM HoloTC Standard Calibrators (4.3mL each). Calibrator A is phosphate buffer with protein (Bovine) stabilizers. Calibrators B-F contains HoloTC in phosphate buffer with protein (Bovine) stabilizers to yield the concentrations (pmol/L) shown below:

- Cal A - 0 pmol/L
- Cal B - 8 pmol/L
- Cal C - 16 pmol/L
- Cal D - 32 pmol/L
- Cal E - 64 pmol/L
- Cal F - 128 pmol/L

Preservative: Sodium Azide.

## Controls:

The AxSYM® HoloTC Controls are for use in quality control to monitor the accuracy and precision of the AxSYM HoloTC assay when used for the quantitative determination of holotranscobalamin (HoloTC) in human serum and plasma on the AxSYM System. Each HoloTC Control kit contains 2 bottles (8.3 mL each) of AxSYM HoloTC Controls containing HoloTC in processed human serum/phosphate buffer to yield the concentrations (pmol/L) as follows:

| Control | HoloTC Concentration (pmol/L) | Range (pmol/L) |
| --- | --- | --- |
| Control Low | 21 | 9 - 33 |
| Control High | 48 | 26 - 70 |

The human serum is nonreactive for HBsAg, HIV-1 Ag or HIV-1 RNA, anti-HIV-1/HIV-2, and anti-HCV or HCV RNA. Preservative: Sodium Azide.

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J. Substantial Equivalence Information:

Axis-Shield AxSYM® HoloTC assay
Predicate: Axis-Shield HoloTC RIA (K030655)

SIMILARITIES between the submission (test) and predicate are:
1. Intended use statements.
2. The operating principles are similar in that they both employ antibody/antigen interactions.
3. Both assays are quantitative.
4. Both assays are for use with human serum and plasma.
5. Both assays report concentrations of analyte in pmol/L

DIFFERENCES between the submission (test) and predicate are:
1. The test is an enzyme immunoassay and the predicate is a radioimmunoassay.
2. The test is a sandwich format; the predicate is a competitive binding format.
3. The test uses specific anti-HoloTC antibody-coated microparticles whereas the predicate device uses anti-TC antibody.
4. The test quantitates holotranscobalamin (vitamin B12 bound to transcobalamin) directly whereas the predicate quantitates the cobalamin that was bound to transcobalamin (in the form of cyanocobalamin).
5. The test is an automated assay; the predicate is a manual assay.

| Parameter | Submission Device
Axis-Shield AxSYM®
HoloTC | Predicate Device
Axis-Shield HoloTC RIA |
| --- | --- | --- |
| 510 (k) No. | k062467 | k030655 |
| Intended use | AxSYM HoloTC assay is a Microparticle Enzyme Immunoassay (MEIA) for the quantitative determination of holotranscobalamin (HoloTC) in human serum and plasma on the AxSYM System. HoloTC is used as an aid in the diagnosis and treatment of vitamin B_{12} deficiency. | The Axis-Shield Radioimmunoassay (RIA) is an in vitro diagnostic assay for the quantitative measurement of holotranscobalamin (vitamin B_{12} bound to transcobalamin) in human serum or plasma. Measurements obtained by this device are used in the diagnosis and treatment of vitamin B_{12} deficiency. |
| Technology Format | Automated.
Sandwich format.
Microparticle Enzyme Immunoassay (MEIA). | Manual.
Competitive binding format.
Radioimmunoassay (RIA). |

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| Capture Antibody | Anti-HoloTC (mouse, monoclonal) antibody coated microparticles. | Anti-TC (mouse, monoclonal) antibody coated microparticles |
| --- | --- | --- |
| Detection Antibody | Anti-TC: Alkaline Phosphatase conjugate. | Not applicable |
| Substrate | 4-Methylumbelliferyl phosphate. | Not applicable |
| Assay End-Point | Fluorescence | Radioactivity |
| Protocol steps/principle of procedure | • Incubate the sample with the anti-HoloTC antibody-coated microparticles.
• Add anti-TC: alkaline phosphatase conjugate and incubate.
• Transfer to matrix cell.
• Wash to remove unbound substances
• Add substrate.
• Measure fluorescent product. | • Incubate the sample with anti-TC antibody coated particles.
• Following separation of the bound TC a reducing reagent (Reductant), denaturing reagent (Extractant) and ^{57}Co labeled vitamin B_{12} (cobalamin) Tracer are added.
• The vitamin B_{12} cobalamin) released from TC competes with the tracer for binding to intrinsic factor (IF)
• Unbound tracer is removed by centrifugation.
• The measured radioactivity bound is inversely proportional to the vitamin B_{12} bound to TC in the sample |

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| Parameter | Submission Device Axis-Shield AxSYM® HoloTC | Predicate Device Axis-Shield HoloTC RIA |
| --- | --- | --- |
| Quantitation | Results are determined from a standard calibration curve (0, 8, 16, 32, 64, 128 pmol/L) generated and stored on the instrument. | Results are determined from a calibration curve (0, 10, 20, 40, 80, 160 pmol/L). |
| Standardization | AxSYM HoloTC calibrators (made from the same recombinant HoloTC as in the RIA) were value assigned (using regression analysis) by running 204 samples in both the RIA and AxSYM. Calibrators in the RIA were prepared gravimetrically from a stock solution of recombinant HoloTC. | Calibrators are prepared gravimetrically from a stock solution of recombinant HoloTC. |
| Standard Calibrator Range | 0-128 pmol/L | 0-160 pmol/L |
| Analytical Sensitivity | ≤1 pmol/L | 6 pmol/L |
| Imprecision | Within run CV of 3.4% to 5.1%. Total CV of 6.3% to 8.5%. | Within run CV of 5% to 10% Total CV of 6% to 12%. |
| Reagent stability | Up to stated expiration date when stored at 2-8°C. | Up to stated expiration date when stored at 2-8°C. |
| Sample stability | 28 days at 2-8°C: store at -20°C or colder if testing will be delayed beyond 28 days. Specimens stored at -20°C or colder for 6 months did not show performance differences. | 7 days at 2-8°C; store frozen if testing is delayed more than 7 days. |
| Expected values | In a representative study: N = 281 healthy subjects (age range 21-70), reference interval = 19.1-119.3 pmol/L (central 95% of population). | In a representative study: N = 303 healthy subjects (age range 22-88), reference interval = 37-171 pmol/L (central 95% of population). |

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# K. Standard/Guidance Document Referenced (if applicable):

| STANDARDS |
| --- |
| Title and Reference Number |
| How to Define and Determine Reference Intervals in the Clinical Laboratory; Approved Guideline - Second Edition (C28-A2) |

Other Standards

| GUIDANCE | | | |
| --- | --- | --- | --- |
| Document Title | Office | Division | Web Page |
| Points to Consider Guidance Document on Assayed and Unassayed Quality Control Material; Draft | OIVD | | http://www.fda.gov/cdrh/ode/99.html |
| Format for Traditional and Abbreviated 510(k) s - Guidance for Industry and FDA Staff | OIVD | | http://www.fda.gov/cdrh/ode/guidance/1567.html |

# L. Test Principle:

The assay is based upon microparticle enzyme immunosaay technology (MEIA). When human HoloTC antigen is present in the sample; it binds to the coated microparticles, forming antigen-antibody complexes on the microparticles. An enzymatic reaction yields a fluorescent product which is measured by the analyzer.

# M. Performance Characteristics (if/when applicable):

1. Analytical performance:

a. Precision/Reproducibility:

Precision studies were done according the National Committee for Clinical Laboratory Standards (NCCLS) Protocol EP5-A2. This study assessed repeatability (within-run precision), reproducibility (total precision based on between-day, between run, within-day and between instrument precision).

Precision of the system was demonstrated by assaying the low and high kit controls for the AxSYM HoloTC assay. Assays were run twice daily for 20 days (leaving at least 2 hours between assay runs) on each of two AxSYM instruments. In the event of a lost day, additional day(s) were added to the study in order to generate 20 days worth of complete data.

A calibration curve was generated on each AxSYM during Run 1 on Day 1,

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calibrators and controls were run as samples in replicates of two (n=80 for each control).

## Results:

Analysis included the with-in run and total SD and %CV. Summary results are shown below.

Precision Summary Statistics, HoloTC Low Control and High Control

| Low Control | | Total | | Within Run | |
| --- | --- | --- | --- | --- | --- |
| AxSYM | Mean (pmol/ L) | SD | %CV | SD | %CV |
| 1 | 23.3 | 1.48 | 6.3 | 1.03 | 4.4 |
| | 22.8 | 1.49 | 6.5 | 0.77 | 3.4 |
| High Control | | Total | | Within Run | |
| --- | --- | --- | --- | --- | --- |
| AxSYM | Mean (pmol/ L) | SD | %CV | SD | %CV |
| 2 | 49.7 | 3.87 | 7.8 | 2.51 | 5.1 |
| | 48.2 | 4.09 | 8.5 | 2.36 | 4.9 |

Precision (both reproducibility and repeatability) met the sponsors established criteria specification of ≤ 10% CV.

## b. Linearity/assay reportable range:

The reported or measurable range of the AxSYM HoloTC assay was defined by the analytical sensitivity and the highest calibrator concentration. The sponsor claims an analytical sensitivity of ≤ 1 pmol/L and a high calibrator concentration of 128pmol/L.

Dilution linearity within the working range of the assay was demonstrated to meet all criteria for linearity (acceptable range is 80% to 120%). Recovery test of HoloTC spiked serum samples was demonstrated to be within acceptance criteria of 80% to 120%. Dilution of three commercially available pools of a high HoloTC sample into six dilutions showed correlation coefficients of: pool 1 = R² = 0.9747 y = 1.0507 + 1.0222; pool 2 = R² = 0.978 y = 0.9613 x + 6.1325; and pool 3 = R² = 0.9948 y = 1.0345x - 0.3515. The above least square regression plot results for diluted AxSYM HoloTC samples demonstrated linearity over the range 19.8 - 103pmol/L.

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

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HoloTC calibrators are prepared gravimetrically from a stock solution of recombinant HoloTC. The purity of the recombinant HoloTC is determined (i) by SDS electrophoresis and (ii) spectroscopically from the ratio between the protein component and the cobalamin component.

HoloTC Controls are prepared from human serum and assigned values with reference to the HoloTC calibrators.

d. Detection limit:

The sponsor claims an analytical sensitivity of ≤ 1pmol/L. The sponsor defined the analytical sensitivity of the AxSYM HoloTC assay as the concentration at two standard deviations above the AxSYM HoloTC Standard Calibrator A (0.0pmol/L) and represents the lowest concentration of HoloTC that can be distinguished from zero. In a representative study, the analytical sensitivity was evaluated by repeated testing (n=12 runs in replicates of 10 across two reagent lots) of the AxSYM HoloTC Standard Calibrator A. In this study, the mean analytical sensitivity was calculated as 0.08pmol/L. (observed range 0.03 to 0.14pmol/L).

e. Analytical specificity:

Serum samples were spiked with potentially interfering substances and tested in the HoloTC assay. the interference was ≤ 10% for all the following:

| Interfering Substance | Pool ID | Replicates tested | Interfering Substance conc. | Control HoloTC conc. (pmol/L) | Supplemented Sample HoloTC conc. (pmol/L) | Mean Interference (%) |
| --- | --- | --- | --- | --- | --- | --- |
| Bilirubin | Low | 3 | 0.4 mg/mL | 20.8 | 19.1 | -8 |
| | High | 3 | | 47.9 | 47.9 | 0 |
| Hemoglobin | Low | 3 | 5 mg/mL | 24.4 | 25.6 | 5 |
| | High | 3 | | 63.2 | 64.4 | 2 |
| Triglycerides | Low | 3 | 15 mg/mL | 25.3 | 25.4 | 0 |
| | High | 3 | | 58.9 | 57.3 | -3 |
| Total Protein | Low | 3 | 95 g/L | 12.9 | 14.2 | 10 |
| | High | 3 | | 37.3 | 36.7 | -2 |

The AxSYM HoloTC assay was also evaluated for analytical specificity in a study where cross-reactivity with B₁₂ binding proteins transcobalamin and haptocorrin were measured by the assay. 500pmol/L transcobalamin was added to line diluent and 5000pmol/L haptocorrin was added to Calibrator A and then assayed. (n = 5 replicates) Data from this study are summarized in the table below.

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| Cross-reactant | Cross-reactant Concentration (pmol/L) | % Cross-reactivity |
| --- | --- | --- |
| Transcobalamin | 500 | Not detected |
| Haptocorrin | 5000 | Not detected |

f. Assay cut-off:

Not applicable

2. Comparison studies:

a. Method comparison with predicate device:

A study was performed comparing the AxSYM HoloTC assay to the predicate Axis Shield HoloTC RIA assay. The results from the Passing-Babcock linear regression analysis are summarized in the following table.

| Passing-Bablok Comparison | | | | | |
| --- | --- | --- | --- | --- | --- |
| n | slope | 95% CI on slope | intercept | 95% CI on intercept | r |
| 204 | 1.02 | 0.95 to 1.10 | 2.1 | -0.91 to 4.84 | 0.90 |

Sample range for RIA: 7.1 – 144.3 pmol/L

Sample range for AxSYM: 8.9 – 123.3 pmol/L

b. Matrix comparison:

Matrix comparison studies to determine which anticoagulants tube types are suitable for collection of samples to be measured with the AxSYM HoloTCC assay was performed. One tube of plasma was collected in each of the following collection tubes from 10 in-house volunteers: serum, Lithium Heparin, serum gel separator and EDTA plasma tubes. The tubes were processed according to the manufacturer’s guidelines and the plasma removed from each tube and stored. Each sample was tested in replicates of 3 for each tube type on one instrument. The comparison results are presented in the table below.

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| Sample No. | Serum (glass) | Lithium Heparin | Serum Gel Separator | EDTA | Overall % Difference from Serum (glass) | | |
| --- | --- | --- | --- | --- | --- | --- | --- |
| | | | | | Lithium Heparin | Serum Gel Separator | EDTA |
| 1 | 25.22 | 26.70 | 24.01 | 31.48 | 5.89 | -4.79 | 24.84 |
| 2 | 25.58 | 25.56 | 23.71 | 34.27 | -0.09 | -7.3 | 33.94 |
| 3 | 30.14 | 26.31 | 23.35 | 37.46 | -12.72 | -22.53 | 24.26 |
| 4 | 31.81 | 31.82 | 30.02 | 44.34 | 0.03 | -5.65 | 39.36 |
| 5 | 31.11 | 35.15 | 31.63 | 43.59 | 12.97 | 1.66 | 40.11 |
| 6 | 37.97 | 39.71 | 34.73 | 55.53 | 4.58 | -8.54 | 46.23 |
| 7 | 36.63 | 40.39 | 33.58 | 57.39 | 10.25 | -8.32 | 56.64 |
| 8 | 19.92 | 21.61 | 18.45 | 27.90 | 8.48 | -7.34 | 40.1 |
| 9 | 23.02 | 23.93 | 21.59 | 31.67 | 3.92 | -6.2 | 37.56 |
| 10 | 65.87 | 60.34 | 57.86 | 94.11 | -8.4 | -12.17 | 42.86 |
| Grand Mean | 32.73 | 33.15 | 29.89 | 45.77 | 2.49 | -812 | 38.59 |

The sponsors established acceptance criteria of grand mean % difference $\leq / \pm 10\%$ to serum was met for Lithium Heparin plasma and gel-separator Serum collection tubes. EDTA Plasma is not recommended for use in this assay.

3. Clinical studies:

a. Clinical Sensitivity:

Not applicable

b. Clinical specificity:

Not applicable

c. Other clinical supportive data (when a. and b. are not applicable):

4. Clinical cut-off:

Not applicable

5. Expected values/Reference range:

In a representative study, serum specimens from 281 healthy donors were tested using the AxSYM HoloTC assay. The mean HoloTC concentration was 47.7 pmol/L with a range from 8.9 to 123.3 pmol/L. The central 95% of the population defined the expected range of 19.1 to 119.3 pmol/L.

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In the labeling the sponsor recommends that each laboratory determine its own reference range based upon its particular locale and population characteristics.

**N. Proposed Labeling:**

The labeling is sufficient and it satisfies the requirements of 21 CFR Part 809.10.

**O. Conclusion:**

The submitted information in this premarket notification is complete and supports a substantial equivalence decision.

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