LIAISON Vitamin B12

K192064 · DiaSorin, Inc. · CDD · Oct 2, 2019 · Clinical Chemistry

Device Facts

• Record ID: K192064
• Device Name: LIAISON Vitamin B12
• Applicant: DiaSorin, Inc.
• Product Code: CDD · Clinical Chemistry
• Decision Date: Oct 2, 2019
• Decision: SESE
• Submission Type: Traditional
• Regulation: 21 CFR 862.1810
• Device Class: Class 2

Indications for Use

The DiaSorin LIAISON® Vitamin B12 assay uses chemiluminescent immunoassay (CLIA) technology for the quantitative determination of Vitamin B12 in human serum, SST serum and lithium heparin plasma. Measurements obtained by this device are used in the diagnosis and treatment of anemia's of gastrointestinal malabsorption. Assay results should be used in conjunction with other clinical or laboratory data to assist the clinician in making individual patient management decisions. The assay must be performed on the LIAISON® XL Analyzer.

Device Story

The LIAISON® Vitamin B12 is a competitive chemiluminescent immunoassay (CLIA) performed on the LIAISON® XL Analyzer. It processes human serum, SST serum, or lithium heparin plasma samples. The assay involves three incubation steps: dissociation of Vitamin B12 from binding proteins; binding of Vitamin B12 to intrinsic factor on magnetic particles; and competition with Vitamin B12 linked to an isoluminol derivative. After washing, starter reagents initiate a flash chemiluminescent reaction. A photomultiplier measures the light signal (Relative Light Units), which is inversely proportional to the Vitamin B12 concentration. Results are used by clinicians to diagnose and manage anemias related to gastrointestinal malabsorption. The device provides quantitative results to assist in clinical decision-making.

Clinical Evidence

Bench testing only. Precision study (N=80) showed total CVs 6.7-10.0%. Linearity confirmed across 55-1500 pg/mL range (R > 0.99). Interference testing followed CLSI EP07-A2; no interference observed for common substances up to specified concentrations. Method comparison (N=155) against predicate yielded Passing-Bablok slope 0.97 (95% CI: 0.94-1.00) and correlation coefficient 0.985. Matrix comparison (N=48) confirmed equivalence between serum, SST serum, and lithium heparin plasma. Reference range study (N=166) established 2.5th-97.5th percentile range of 107.2-653.3 pg/mL.

Technological Characteristics

Competitive chemiluminescent immunoassay (CLIA). Solid phase: magnetic particles coated with intrinsic factor. Conjugate: polymer conjugated with Vitamin B12 and isoluminol derivative. Energy source: flash chemiluminescent reaction measured by photomultiplier. Analytical measuring range: 55-1500 pg/mL. Sample volume: 100 μL. Requires LIAISON® XL Analyzer. Reagents include buffers (glycine, potassium hydroxide, potassium phosphate) and stabilizers. Standards referenced: CLSI EP05-A3, EP15-A3, EP07-A2, EP06-A, EP17-A2, EP28-A3C.

Indications for Use

Indicated for the quantitative determination of Vitamin B12 in human serum, SST serum, and lithium heparin plasma to assist in the diagnosis and treatment of anemias of gastrointestinal malabsorption. For use by clinicians in conjunction with other clinical or laboratory data.

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

• Beckman Coulter Access Vitamin B12 assay (K140496)

Submission Summary (Full Text)

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Food and Drug Administration
10903 New Hampshire Avenue
Silver Spring, MD 20993-0002
www.fda.gov

# 510(k) SUBSTANTIAL EQUIVALENCE DETERMINATION DECISION SUMMARY

ASSAY ONLY

## I Background Information:

A 510(k) Number
K192064

B Applicant
DiaSorin Inc.

C Proprietary and Established Names
LIAISON® Vitamin B12

D Regulatory Information
| Product Code(s) | Classification | Regulation Section | Panel |
| --- | --- | --- | --- |
| CDD | Class II | 21 CFR 862.1810 - Vitamin B12 Test System | CH - Clinical Chemistry |

## II Submission/Device Overview:

A Purpose for Submission:
New Device

B Measurand:
Vitamin B12

C Type of Test:
Quantitative, chemiluminescent immunoassay

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## III Intended Use/Indications for Use:

### A Intended Use(s):

See Indications for Use below.

### B Indication(s) for Use:

The DiaSorin LIAISON® Vitamin B12 assay uses chemiluminescent immunoassay (CLIA) technology for the quantitative determination of Vitamin B12 in human serum, SST serum and lithium heparin plasma. Measurements obtained by this device are used in the diagnosis and treatment of anemia's of gastrointestinal malabsorption. Assay results should be used in conjunction with other clinical or laboratory data to assist the clinician in making individual patient management decisions.

The assay must be performed on the LIAISON® XL Analyzer.

### C Special Conditions for Use Statement(s):

Rx - For Prescription Use Only

For in vitro diagnostic use only

### D Special Instrument Requirements:

LIAISON® XL Analyzer

## IV Device/System Characteristics:

### A Device Description:

The LIAISON® Vitamin B12 reagent contains:

- Magnetic particles - coated with Intrinsic Factor in phosphate buffer, stabilizers and preservatives; 2.4 mL
- Calibrator 1 - Vitamin B12 free human serum containing cyanocobalamin (Vitamin B12), and preservatives; 2.4 mL
- Calibrator 2 - Vitamin B12 free human serum containing cyanocobalamin (Vitamin B12), and preservatives; 2.4 mL
- Conjugate - Proprietary polymer conjugated with Vitamin B12 and an isoluminol derivative, in MES buffer with surfactant and preservatives; 12 mL
- Assay Buffer 1 – Glycine buffer containing TCEP (tris(2-carboxyethyl) phosphine) and preservatives; 3.0 mL
- Assay Buffer 2 – Potassium hydroxide buffer containing 0.05% potassium cyanide and preservatives; 12 mL

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- Assay Buffer 3 – Potassium phosphate with dicyanocobinimide, surfactant and preservatives; 22 mL

## B Principle of Operation:

The LIAISON® Vitamin B12 assay is a competitive chemiluminescence immunoassay (CLIA) for quantitative determination of Vitamin B12 in serum, SST serum and lithium heparin plasma. During the first incubation, Vitamin B12 is dissociated from its binding protein. After the initial incubation of 10.5 minutes, Vitamin B12 binds to an intrinsic factor on the solid phase. After a second incubation of 10.5 minutes, a Vitamin B12 linked to an isoluminol derivative is added to compete with the Vitamin B12 in the sample. After a third incubation of 5.25 minutes, the unbound material is removed with a wash cycle. Subsequently, the starter reagents are added to initiate a flash chemiluminescent reaction. The light signal is measured by a photomultiplier as relative light units (RLU) and is inversely proportional to the concentration of Vitamin B12 present in samples.

## V Substantial Equivalence Information:

### A Predicate Device Name(s):

Access Vitamin B12 Assay

### B Predicate 510(k) Number(s):

K140496

### C Comparison with Predicate(s):

| Device & Predicate Device(s): | K192064 | K140496 |
| --- | --- | --- |
| Device Trade Name | LIAISON® Vitamin B12 | Beckman Coulter Access Vitamin B12 |
| General Device Characteristic Similarities | | |
| Intended Use/Indications For Use | For the quantitative determination of Vitamin B12 in serum and plasma. | Same |
| Assay Type | Chemiluminescence immunoassay | Same |
| Results | Quantitative | Same |
| Operation | Automated | Same |
| General Device Characteristic Differences | | |
| Sample Type | Human serum, serum-separating tubes (SST) | uman serum and |

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| Device & Predicate Device(s): | K192064 | K140496 |
| --- | --- | --- |
| | and lithium heparin plasma | heparin plasma |
| Sample Size | 100 μL | 45 μL |
| Reagent Storage | 2-8°C | 2-10 °C |
| Analytical Measuring Range | 55-1500 pg/mL | 50-1500 pg/mL |
| Solid Phase | Magnetic particles coated intrinsic factor | Paramagnetic particles coated with goat anti-mouse IgG |
| Conjugate | Polymer conjugated with Vitamin B12 | Porcine intrinsic factor-alkaline phosphate (bovine) conjugate |

VI Standards/Guidance Documents Referenced:

- CLSI Guideline EP05-A3, Evaluation of Precision of Quantitative Measurement Procedures; Approved Guideline.
- CLSI Guideline EP15-A3, User Verification of Precision and Estimation of Bias; Approved Guideline.
- CLSI Guideline EP07-A2, Interference Testing in Clinical Chemistry, Approved Guideline.
- CLSI Guideline EP06-A, Evaluation of the Linearity of Quantitative Measurement Procedures; A Statistical Approach; Approved Guideline.
- CLSI Guideline EP 17-A2, Evaluation of Detection Capability for Clinical Laboratory Measurement Procedures; Approved Guideline.
- CLSI Guideline EP28-A3C, Defining, Establishing and Verifying Reference Intervals in the Clinical Laboratory; Approved Guideline.

VII Performance Characteristics (if/when applicable):

A Analytical Performance:

1. Precision/Reproducibility:

A 20-day reproducibility/precision study was performed following CLSI EP05-A3 guideline. Testing was performed using a coded panel comprised of 6 serum samples, spanning the assay range was tested, along with the LIAISON® Vitamin B12 kit controls (2 levels). Each

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sample was tested in duplicate per run in two separate runs per day, over 20 operating days $(\mathrm{N} = 80)$ . The results are summarized in the below table.

| Sample ID | Mean (pg/mL) | ntra-Run Precision | | Total Precision | |
| --- | --- | --- | --- | --- | --- |
| | | SD | %CV | SD | %CV |
| Control 1 | 642 | 21.56 | 3.4% | 42.99 | 6.7% |
| Control 2 | 1262 | 48.79 | 3.9% | 86.28 | 6.8% |
| Serum #1 | 240 | 10.16 | 4.2% | 20.36 | 8.5% |
| Serum #2 | 417 | 21.85 | 5.2% | 39.76 | 9.5% |
| Serum #3 | 646 | 24.83 | 3.8% | 57.55 | 8.9% |
| Serum #4 | 773 | 29.36 | 3.8% | 60.12 | 7.8% |
| Serum #5 | 1039 | 39.87 | 3.8% | 103.87 | 10.0% |
| Serum #6 | 1093 | 47.96 | 4.4% | 93.89 | 8.6% |

# 2. Linearity:

One sample of each specimen type (serum, SST serum, lithium heparin plasma) containing endogenous and/or spiked Vitamin B12 above the measuring range of the assay at $1500~\mathrm{pg / mL}$ was diluted with a low-level sample to create 13 evenly distributed concentrations spanning the measuring range $(< 50 - > 1500~\mathrm{pg / mL})$ . Each sample was tested in duplicate following CLSI EP06-A guideline. The results for each sample were analyzed by regression of observed concentration (y) versus expected concentration (x). The regression results are summarized below for each sample type:

Serum: $y = 1.076x + 6.896$ ; $R = 0.998$

SST Serum: $\mathrm{y} = 1.009\mathrm{x} + 0.224$ . $\mathrm{R} = 0.999$

Lithium Heparin plasma: $\mathrm{y} = 1.029\mathrm{x} - 0.095$ . $\mathrm{R} = 0.996$

# 3. Analytical Specificity/Interference:

Interference testing was conducted following CLSI-EP07-A2 guideline. Potential interfering substances were spiked to serum samples with normal and high level of Vitamin B12 (200 and $800~\mathrm{pg / mL}$ ). The same samples without interferent but containing the "vehicle" were tested as controls. Interference was defined as more than $10\%$ difference between the spiked and the un-spiked samples.

The substances and the highest concentrations tested without interference are summarized in the below table:

| Drug/Substance | Concentration Tested |
| --- | --- |
| Hemoglobin | 300 mg/dL |
| Bilirubin (conjugated) | 40 mg/dL |
| Bilirubin (unconjugated) | 40 mg/dL |
| Triglycerides | 3,000 mg/dL |
| Cholesterol | 500 mg/dL |
| Albumin | 12 g/dL |
| Human IgG | 12 g/dL |

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| Drug/Substance | Concentration Tested |
| --- | --- |
| HAMA | 1387.5 ng/mL |
| Rheumatoid Factor | 1035 IU/mL |
| Acetaminophen | 20 mg/dL |
| Acetylsalicylic Acid | 65 mg/dL |
| Ibuprofen | 50 mg/dL |
| Biotin | 2 mg/mL |

Under the limitations of the procedure in the labeling, the sponsor listed the substances that are known to interfere with the assay:

- Heterophilic antibodies in human serum can react with reagent immunoglobulins or other reagent material, interfering with in vitro immunoassays.
- Patients routinely exposed to animals, animal serum products, or other immunogenic products that may elicit heterophilic antibody production against the assay’s reagents can be prone to this interference and anomalous values may be observed.
- Approximately 50% of patients with pernicious anemia have Intrinsic factor antibodies. During the reaction, the denaturation should inactivate intrinsic factor blocking antibodies. However, any remaining anti-intrinsic factor antibody or extremely high titer of the intrinsic factor antibody could cause erroneous results.

## Cross-reactivity

Cross-reactivity was evaluated following CLSI-EP07-A2 guideline. The % cross reactivity is calculated as follows:

% cross reactivity = (Corrected Assay Value/ Cross-reactant Concentration) * 100.

The result is summarized in the below table.

| Cross-Reactant | Spiked Concentration | % Cross Reactivity |
| --- | --- | --- |
| Dicyanocobinamide | 10,000pg/mL | -0.19% |

## 4. Assay Reportable Range:

55-1500 pg/mL

## 5. Traceability, Stability, Expected Values (Controls, Calibrators, or Methods):

**Traceability**

The LIAISON® Vitamin B12 Calibrators are traceable to an in-house standard preparation of Vitamin B12 (pg/mL).

## 6. Detection Limit:

FTo determine the limit of blank (LoB), five blank samples were tested using 2 lots of reagent on 1 analyzer by two operators. Each sample was run in 2 replicates /run for 6 runs on three days yielding 60 results per reagent lot.

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LoB was calculated as: $\mathrm{LoB} = \mu (\mathrm{Blank}) + 1.653*\sigma (\mathrm{Blank})$

FThe limit of detection (LoD), was determined by testing four samples in the range of approximately 25 to $100\mathrm{pg / mL}$ on 2 analyzers using two reagent lot by two operators. Each sample was tested in 2 replicates per run for over 6 runs over 3 days (yielding a minimum of 96 results).

LoD was calculated for each lot as: $\mathrm{LoD} = \mathrm{LoB} + 1.655*\sigma s$ (Low Sample)

For limit of quantitation (LoQ), eight samples (4 of which were used in the LoD determination) in the range of 25 to approximately $250~\mathrm{pg / mL}$ were tested using 2 reagent lots on 2 analyzers by 2 operators. Each sample was tested in 2 replicates per run for 6 runs over 3 days, yielding a minimum of 144 results. LoQ was defined as the lowest concentration at which the regression line crosses the $20\%$ CV line and was calculated for each of the reagent lots using a curve fit best suited to the data set.

The claimed LoB, LoD and LoQ are summarized in the below table using the highest concentration resulted from the different lots tested.

| LoB | LoD | LoQ |
| --- | --- | --- |
| 38.7 pg/mL | 51.2 pg/mL | 55 pg/mL |

# 7. Assay Cut-Off:

Not applicable.

# B Comparison Studies:

# 1. Method Comparison with Predicate Device:

method comparison study was performed using a total of 156 serum samples (147 native, 9 spiked). The samples were tested in singlicate using the candidate device and predicate device, and the results were compared.

One sample was removed from the analysis as it was below the measuring range of the assay. The samples ranged from 71.2 - 1315 pg/mL pg/mL.

The result of the Passing-Bablok regression analysis is summarized in the below table:

| N | Slope (95% CI) | Intercept (95% CI) | Correlation Coefficient |
| --- | --- | --- | --- |
| 155 | 0.97 (0.94-1.00) | 6.25 (-3.50-18.29) | 0.985 |

# 2. Matrix Comparison:

Forty-eight (48) matched patient sets of serum, SST serum and lithium heparin plasma samples were tested to determine if these sample types provide equivalent results on the LIAISON® Vitamin B12 assay.

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The results of the regression analysis are summarized below:

SST serum compared to Serum

| N | Slope (95% CI) | Intercept (95% CI) | Correlation Coefficient |
| --- | --- | --- | --- |
| 48 | 0.97 (0.94-0.99) | 8.21 (2.14-16.56) | 0.996 |

Lithium Heparin compared to Serum

| N | Slope (95% CI) | Intercept (95% CI) | Correlation Coefficient |
| --- | --- | --- | --- |
| 48 | 1.10 (1.05 to 1.15) | 14.78 (1.09-29.87) | 0.988 |

The study results support the sponsor's claim that human serum and lithium heparin plasma are acceptable sample types to be used with this assay.

## C Clinical Studies:

1. Clinical Sensitivity:

Not applicable.

2. Clinical Specificity:

Not applicable.

3. Other Clinical Supportive Data (When 1. and 2. Are Not Applicable):

None.

## D Clinical Cut-Off:

Not applicable.

## E Expected Values/Reference Range:

A prospective reference range study was performed with serum samples from 166 apparently healthy adults aged 21 - 59 years of age from mixed ethnic backgrounds (30% Caucasian, 31% African Americans, 39% Hispanics) who were fasting for at least 8 hrs. Apparently healthy status was defined as subjects who had no history of anemias, folic acid or B12 deficiency, inflammatory bowel disease (IBD) or suspected IBD, celiac disease, gastrointestinal malabsorption disorders, or eating disorders. Inclusion criteria also include no oral contraceptive use within 3 months, and no alcohol within 48 hrs of blood draw or excessive alcohol usage. Pregnant women or anyone taking B12 supplement were excluded from the study.

Based on the 95% confidence interval, the following values were established following CLSI guideline EP28-A3c.

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Observed Reference Ranges

| Population (n=166) | Median | Observed Range 2.5^{th} to 97.5^{th} Percentile |
| --- | --- | --- |
| United States | 318.5 (pg/mL) | 107.2 pg/mL – 653.3 pg/mL |

The sponsor recommended in the labeling that each laboratory establish its own range of expected values for the population taken into consideration.

## VIII Proposed Labeling:

The labeling supports the finding of substantial equivalence for this device.

## IX Conclusion:

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