VITROS CHEMISTRY PRODUCTS HCY REAGENT, VITROS CHEMISTRY PRODUCTS CALIBRATOR KIT 27, HCY PERFORMANCE VERIFIERS

K061588 · Ortho-Clinical Diagnostics, Inc. · LPS · Jul 21, 2006 · Clinical Chemistry

Device Facts

• Record ID: K061588
• Device Name: VITROS CHEMISTRY PRODUCTS HCY REAGENT, VITROS CHEMISTRY PRODUCTS CALIBRATOR KIT 27, HCY PERFORMANCE VERIFIERS
• Applicant: Ortho-Clinical Diagnostics, Inc.
• Product Code: LPS · Clinical Chemistry
• Decision Date: Jul 21, 2006
• Decision: SESE
• Submission Type: Traditional
• Regulation: 21 CFR 862.1377
• Device Class: Class 2

Indications for Use

VITROS Chemistry Products HCY Reagent: For in vitro diagnostic use only. VITROS Chemistry Products HCY Reagent is used to quantitatively measure total homocysteine (HCY) concentration in human serum and plasma. Serum and plasma homocysteine levels can assist in the diagnosis and treatment of patients suspected of having hyperhomocysteinemia and homocystinuria. VITROS Chemistry Products Calibrator Kit 27: For in vitro diagnostic use only. VITROS Chemistry Products Calibrator Kit 27 is used to calibrate VITROS 5,1 FS Chemistry Systems for the quantitative measurement of homocysteine (HCY). VITROS Chemistry Products HCY Performance Verifiers I, II & III: For in vitro diagnostic use only. VITROS Chemistry Products HCY Performance Verifiers are assayed controls used to monitor performance of VITROS HCY Reagents on VITROS 5,1 FS Chemistry Systems.

Device Story

VITROS Chemistry Products HCY Reagent, Calibrator Kit 27, and Performance Verifiers are used on VITROS 5,1 FS Chemistry Systems to measure total homocysteine in human serum/plasma. Disulfide-linked homocysteine is reduced by TCEP; reduced homocysteine reacts with serine via cystathionine β-synthase (CBS) to form L-cystathionine. L-cystathionine is cleaved by cystathionine β-lyase (CBL) to produce homocysteine, pyruvate, and ammonia. Pyruvate is reduced to lactate by lactate dehydrogenase (LDH) using NADH. NADH conversion to NAD is measured spectrophotometrically at 340 nm; concentration is directly proportional to NADH conversion. System uses stored calibration curves to determine sample concentration. Used in clinical laboratory settings by trained personnel. Results assist clinicians in diagnosing and treating hyperhomocysteinemia and homocystinuria.

Clinical Evidence

No clinical diagnostic studies performed. Analytical performance established via bench testing: precision (within-lab CV 1.1-3.4%), linearity (0.5-71.0 μmol/L), and method comparison against predicate (n=93, y=0.98x + 1.0, r=0.97). Reference intervals established using 205 healthy adults (106 female, 99 male). Interference testing conducted for various endogenous and exogenous substances.

Technological Characteristics

Homogeneous enzymatic assay; spectrophotometric detection at 340 nm. Reagents: TCEP, CBS, CBL, LDH. Calibrators: aqueous solution with amino acids/inorganic acid. Controls: processed human serum. Instrumentation: VITROS 5,1 FS Chemistry System. Connectivity: System-integrated. Storage: 2-8°C.

Indications for Use

Indicated for the quantitative measurement of total homocysteine in human serum and plasma to assist in the diagnosis and treatment of patients suspected of having hyperhomocysteinemia and homocystinuria.

Regulatory Classification

Identification

A urinary homocystine (nonquantitative) test system is a device intended to identify homocystine (an analogue of the amino acid cystine) in urine. The identification of urinary homocystine is used in the diagnosis and treatment of homocystinuria (homosystine in urine), a heritable metabolic disorder which may cause mental retardation.

Predicate Devices

• Bio-Rad Homocysteine by HPLC Test (k993107)
• VITROS Chemistry Products AAT Performance Verifiers I, II, & III (k052819)

Submission Summary (Full Text)

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

A. 510(k) Number:
k061588

B. Purpose for Submission:
New device

C. Measurand:
Homocysteine

D. Type of Test:
Quantitative, homogeneous enzymatic assay

E. Applicant:
Ortho-Clinical Diagnostics, Inc.

F. Proprietary and Established Names:
VITROS Chemistry Products HCY Reagent, VITROS Chemistry Products Calibrator Kit 27, VITROS HCY Performance Verifiers I, II, and III

G. Regulatory Information:

| Product Code | Classification | Regulation Section | Panel |
| --- | --- | --- | --- |
| Urinary Homocysteine (Nonquantitative) Test System (LPS) | Class II | 21 CFR 862.1377, Urinary homocysteine (nonquantitative) test system. | 75 Clinical Chemistry (CH) |
| Product Code | Classification | Regulation Section | Panel |
| Calibrators, Secondary (JIT) | Class II | 21 CFR 862.1150, Calibrator | 75 Clinical Chemistry (CH) |
| Product Code | Classification | Regulation Section | Panel |
| Single (Specified) analyte controls (assayed and unassayed) (JJX) | Class I | 21 CFR 862.1660, Quality Control Material (assayed and unassayed) | 75 Clinical Chemistry (CH) |

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H. Intended Use:

1. Intended use(s):

VITROS Chemistry Products HCY Reagent: For in vitro diagnostic use only. VITROS Chemistry Products HCY Reagent is used to quantitatively measure total homocysteine (HCY) concentration in human serum and plasma. Serum and plasma homocysteine levels can assist in the diagnosis and treatment of patients suspected of having hyperhomocysteinemia and homocystinuria.

VITROS Chemistry Products Calibrator Kit 27: For in vitro diagnostic use only. VITROS Chemistry Products Calibrator Kit 27 is used to calibrate VITROS 5,1 FS Chemistry Systems for the quantitative measurement of homocysteine (HCY).

VITROS Chemistry Products HCY Performance Verifiers I, II & III: For in vitro diagnostic use only. VITROS Chemistry Products HCY Performance Verifiers are assayed controls used to monitor performance of VITROS HCY Reagents on VITROS 5,1 FS Chemistry Systems.

2. Indication(s) for use:
See Intended Use above.

3. Special conditions for use statement(s):
Prescription use only

4. Special instrument requirements:
The device is intended for use with the VITROS 5,1 FS Chemistry System. The VITROS 5,1 FS Chemistry System was cleared for market use in k031924.

I. Device Description:

The VITROS Chemistry Products HCY Reagent consists of two dual chambered reagent packs containing three ready-to-use liquid reagents. VITROS Chemistry Products FS Diluent Pack 2 (Saline/BSA) is a common reagent that is used by multiple assays on the VITROS 5,1 FS Chemistry System. This is a dual chambered package containing two ready-to-use liquid diluents. Diluent 1 is prepared from processed water to which inorganic salt has been added. Diluent 2 is prepared from processed water to which bovine serum albumin, inorganic salts and preservatives have been added. Diluent 1 contains saline (154 mM NaCl), which is used to dilute elevated homocysteine (HCY) samples on the analyzer.

VITROS Chemistry Products Calibrator Kit 27 is a two level fluid prepared from an aqueous solution containing amino acids and inorganic acid. These standards are used

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to calibrate VITROS 5,1 FS Chemistry Systems for the quantitative measurement of homocysteine (HCY).

VITROS HCY Performance Verifiers I, II and III are prepared from processed human serum to which amino acid and preservative have been added. These are assayed controls used to monitor performance of VITROS HCY Reagent on VITROS 5,1 FS Chemistry Systems.

J. Substantial Equivalence Information:

| Predicate | k993107 Bio-Rad Homocysteine by HPLC Test |
| --- | --- |
| Describe the item being compared | |
| The Bio-Rad Homocysteine by HPLC test is based on precolumn derivatization and a 5 minute chromatography. The Bio-Rad Homocysteine by HPLC test is intended for the quantitative determination of total homocysteine in human plasma or serum. | |

Device Similarities

| Device Characteristic | VITROS HCY (New device)
BIO-RAD HOMOCYSTEINE by HPLC test (Predicate device) |
| --- | --- |
| Intended use | For in-vitro diagnostic use. Quantitative determination of total homocysteine in human serum or plasma |
| Analyte measured | Homocysteine |
| Sample type | Serum or Plasma |
| Measurement | Quantitative |

Device Differences

| Device Characteristics | VITROS HCY (New device) | BIO-RAD HOMOCYSTEINE by HPLC test (Predicate device) |
| --- | --- | --- |
| Reportable range | 1.0 – 50.0 μmol/L | 0.5 – 100 μmol/L |
| Sensitivity | 1.0 μmol/L | 0.5 μmol/L |
| Calibrator levels | Two levels (0, 27 μmol/L) | Single level (15-20 μmol/L) |
| Calibrator format | Liquid | Lyophilized |
| Calibrator matrix | Aqueous solution containing amino acids and inorganic acid | Human Serum |
| Instrumentation | Automated clinical chemistry analyzer | Isocratic HPLC System |

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| Reference Interval | Males: 6.6 – 14.8 μmol/L
Females: 4.7 – 12.6 μmol/L | < 15 μmol/L |
| --- | --- | --- |
| Method | Homogeneous Enzymatic | Chromatography |
| Predicate | k052819 VITROS Chemistry Products AAT
Performance Verifiers I, II, & III |
| --- | --- |

Describe the item being compared

The VITROS Chemistry Products AAT Performance Verifiers I, II and III are prepared from processed human serum to which inorganic salts, buffers, and preservatives have been added. These are assayed controls used to monitor performance of VITROS AAT Reagent on VITROS 5,1 FS Chemistry Systems.

Device Similarities

| Indications for use | Assayed controls used to monitor assay performance on VITROS 5,1 FS Chemistry Systems |
| --- | --- |
| Matrix | Prepared from processed human serum with preservatives added. |
| Product type | Assayed control |
| Format | Liquid |
| Number of levels | Three |

Device Differences

| Device Characteristic | VITROS HCY Performance Verifiers (new device) | VITROS AAT Performance Verifiers (predicate device) |
| --- | --- | --- |
| Analyte measure | Homocysteine | alpha 1-Antitrypsin (AAT) |

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

STANDARDS

Title and Reference Number

Evaluation of Precision Performance of Clinical Chemistry Devices; Approved Guideline (EP5-A)

Interference Testing in Clinical Chemistry; Approved Guideline (EP 7-A)

Method Comparison and Bias Estimation Using Patient Samples; Approved Guideline (EP09-A2)

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

Other Standards

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| None | | | |
| --- | --- | --- | --- |
| GUIDANCE | | | |
| Document Title | Office | Division | |
| None | | | |

## L. Test Principle:

The quantitative measurement of homocysteine (HCY) is performed using the VITROS Chemistry Products HCY Reagent in conjunction with the VITROS Chemistry Products Calibrator Kit 27 and on the VITROS 5,1 FS Chemistry Systems. The VITROS Chemistry Products HCY Reagent consists of two dual chambered reagent packs containing three ready-to-use liquid reagents. Disulfide linked homocysteine (oxidized forms) in the sample is reduced by Tris (2-Carboxyethyl) phosphine hydrochloride (TCEP) to form reduced homocysteine. Reduced homocysteine reacts with serine in the presence of cystathionine b-synthase (CBS) to form L-cystathionine. L-cystathionine is broken down by cystathionine b-lyase (CBL) to produce homocysteine, pyruvate and ammonia. Pyruvate is reduced to lactate by lactate dehydrogenase (LDH) using NADH as coenzyme. The concentration of homocysteine is directly proportional to the amount of NADH converted to NAD⁺ and is measured spectrophotometrically at 340 nm. Once a calibration has been performed, the homocysteine concentration in each unknown sample can be determined using the stored calibration curve and the measured absorbance obtained in the assay of the sample.

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

### 1. Analytical performance:

#### a. Precision/Reproducibility:

Precision was evaluated with quality control materials and a human serum pool on the VITROS 5,1 FS Chemistry System following NCCLS Protocol EP5.

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VITROS Chemistry Products HCY Reagent Precision (Serum)

| System | SI Units (μmol/L) | | | Within-Lab %CV | No. Days | No. Obser |
| --- | --- | --- | --- | --- | --- | --- |
| VITROS 5,1 FS | Mean Conc. (μmol/L) | Within-Day SD* | Within-Lab SD** | | | |
| | 7.0 | 0.21 | 0.24 | 3.4 | 20 | 80 |
| | 11.9 | 0.23 | 0.27 | 2.3 | 20 | 80 |
| | 41.8 | 0.33 | 0.44 | 1.1 | 20 | 80 |
| | 23.7 | 0.24 | 0.30 | 1.3 | 20 | 80 |
| | 48.0 | 0.52 | 0.66 | 1.4 | 20 | 80 |

*Within Day precision was determined using two-runs/ day with two replicates per run
** Within Lab precision was determined using a single lot of reagents on a single analyzer, calibrating once a week (4 calibrations).

b. Linearity/assay reportable range:

The evaluation of the linearity of the VITROS HCY assay was performed based on NCCLS EP6-A. Three lots of VITROS Chemistry Products HCY Reagents were tested using a series of admixtures with concentrations of 0.50 μmol/L to 71.0 μmol/L. The VITROS Chemistry Products HCY Reagent linear range was determined to be 0.50 μmol/L to 71.0 μmol/L and the reportable range was chosen by the applicant to be 1.0 μmol/L to 50.0 μmol/L.

An on-board dilution study of patient samples with saline was performed using 10 samples with a dilution factor of 2 and 10 samples with a dilution factor of 3. The recovery ranged from 96.9% to 100.0% using the dilution factor of 2 and ranged from 92.4% to 98.5% using the dilution factor of 3.

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

**Traceability**

Both the Calibrators and Verifiers are value assigned by performing multiple determinations using the VITROS 5,1 FS Chemistry Systems and the VITROS Chemistry Products HCY Reagent. The VITROS Chemistry Products Calibrator Kit 27 is traceable to NIST SRM 1955.

**Stability**

The long term (shelf life) stability of the VITROS HCY reagents, calibrators and verifiers were tested on a VITROS 5,1 FS Chemistry System using a real time study extending at least one month beyond the selected expiration date. Testing was done at intervals of 0, 1, 2, 3 months and is still ongoing. The

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VITROS HCY reagents, calibrators and verifiers are stored at refrigerated (2- $8^{\circ}\mathrm{C}$ ).

The opened vial (in use) stability of the VITROS HCY reagents, calibrators and verifiers were tested on a VITROS 5,1 FS Chemistry System. Testing was done at intervals of 0, 7, 14 days and is still ongoing.

# d. Detection limit:

The LOB specimen tested was a homocysteine-free serum pool obtained from Golden West Biologics Inc, Temecula, CA. The LOD specimen tested was consisted of two homocysteine serum pools spiked with homocysteine to nominal concentrations of 0.01 and $0.48\mu \mathrm{mol} / \mathrm{L}$ . The Limit of Blank (LOB), Limit of Detection (LOD), and Limit of Quantitation (LOQ) were determined following NCCLS EP17-A. The lower linear range of the VITROS HCY assay was determined to be $0.5\mu \mathrm{mol} / \mathrm{L}$ based on the results of the linearity evaluation. The value that will be the claimed analytical limit (reportable range) at low levels is $1.0\mu \mathrm{mol} / \mathrm{L}$ based on linearity, LOB, LOD, and LOQ evaluations.

# e. Analytical specificity:

The specificity of the VITROS HCY reagent was evaluated by testing the following substances at homocysteine concentrations of approximately 15.0 to $38.0~\mu \mathrm{mol / L}$ using protocols based on NCCLS Protocol EP7-A and found not to interfere, bias $&lt; 13.6\%$ , at the concentrations shown below.

| Compound | Concentration | |
| --- | --- | --- |
| Acetaminophen | 20 mg/dL | Acetaminophen |
| N-Acetyl L-Cysteine | 326 μg/L | N-Acetyl L-Cysteine |
| Adenosine | 6.7 g/dL | Adenosine |
| Amoxicillin | 20 μg/mL | Amoxicillin |
| Ampicillin | 100 mg/dL | Ampicillin |
| Ascorbic Acid (L) | 10 mg/dL | Ascorbic Acid (L) |
| Aspirin | 50 mg/dL | Aspirin |
| Atorvastatin | 300 ng/mL | Atorvastatin |
| Bilirubin | 30 mg/dL | Bilirubin |
| Caffeine | 10 mg/dL | Caffeine |
| Clopidogrel hydrogensulfate | 50 μg/mL | Clopidogrel hydrogensulfate |
| Creatinine | 30 mg/dL | Creatinine |
| L-Cysteine | 2.4 g/dL | L-Cysteine |

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| Compound | Concentration | |
| --- | --- | --- |
| Doxycyclin | 5 mg/dL | Doxycyclin |
| D-penicillamine | 8 μg/mL | D-penicillamine |
| Enalapril Maleate | 120 ng/mL | Enalapril Maleate |
| Gemfibrozil | 50 μg/mL | Gemfibrozil |
| Gentamycin Sulfate | 120 μg/mL | Gentamycin Sulfate |
| Glutathione | 0.6 g/L | Glutathione |
| DL-Homocysteine thiolactone | 0.77 mg/dL | DL-Homocysteine thiolactone |
| Hemoglobin | 500 mg/dL | Hemoglobin |
| Hydrochlorothiazide | 2 μg/mL | Hydrochlorothiazide |
| Ibuprofen | 40 mg/dL | Ibuprofen |
| Intralipid | 500 mg/dL | Intralipid |
| L-Methionine | 29.8 mg/dL | L-Methionine |
| S-(5.-Adenosyl)-L-Methionine p-toluene sulfonate | 79.9 mg/dL | S-(5.-Adenosyl)-L-Methionine p-toluene sulfonate |
| Metronidazole | 20 mg/dL | Metronidazole |
| Nicotine | 2 mg/dL | Nicotine |
| Nifedipine | 200 ng/mL | Nifedipine |
| Procainamide | 100 μg/mL | Procainamide |
| Propanolol | 5 μg/mL | Propanolol |
| Protein (Serum) | 9.3 g/dL | Protein (Serum) |
| Pyruvic Acid | 17.6 mg/dL | Pyruvic Acid |
| Rheumatoid Factor | 373 IU/mL | Rheumatoid Factor |
| Theophylline | 250 μg/mL | Theophylline |
| Triglycerides | 908 mg/dL | Triglycerides |
| Urea | 500 mg/dL | Urea |
| Warfarin | 10 μg/mL | Warfarin |

Known Interferences

- Patient samples containing cystathionine (reference interval: 0.065 to 0.301 $\mu$mol/L) show positive equimolar interference with the VITROS Chemistry

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Products HCY assay. For example, a sample containing 10.0 μmol/L homocysteine and 0.3 μmol/L cystathionine will generate a VITROS HCY result of 10.3 μmol/L. Elevated cystathionine levels can be observed in patients with certain conditions, such as renal disease, folate deficiency, and cystathionine beta-lyase deficiency.

- For samples that generate a Sample Integrity T-index flag, refer to the Sample Dilution section in the package insert.

## Other Limitations

Results should be interpreted with caution. Certain drugs and clinical conditions are known to alter homocysteine concentration *in vivo*. For example, patients who are taking methotrexate, carbamazepine, phenytoin, nitrous oxide, anticonvulsants, or 6-azuridine triacetate, may have higher levels of homocysteine due to metabolic interference with homocysteine metabolism. For additional information, refer to one of the published summaries cited in the package insert.

f. Assay cut-off:

None

2. Comparison studies:

a. Method comparison with predicate device:

A method comparison study was performed on 93 human serum samples on the VITROS 5,1 FS Chemistry Systems and a commercially available method, based on NCCLS Protocol EP9. Three reagent lots were used for the testing and all samples were analyzed in triplicate. A least squares linear regression analysis demonstrated the following relationship: y=0.98 x + 1.0 μmol/L, with a correlation coefficient = 0.97 where y= results obtained using the VITROS HCY assay and x= results obtained with the commercially available system BIO-RAD homocysteine by HPLC assay.

b. Matrix comparison:

Matrix comparison studies were performed by collecting fresh whole blood from a total of 95 donors. Samples were collected into plain glass, lithium-heparin plasma, K2EDTA plasma, K3EDTA plasma, and serum separator tubes at variable fill volumes and pairings and then centrifuged and the serum separated within 1 hr of collection. For each test condition (specimen type or stability), assay value difference were calculated from the baseline (serum in plain glass for specimen collection and fresh sample for stability). The mean and range of the % differences, across all samples, for each condition were calculated. For each storage condition, means were calculated and the bias from fresh results was compared to the applicant’s acceptance criteria for

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determination of acceptability. All the specimen types tested were acceptable at the specimen matrix and stability claims except the plasma samples must be collected in plasma tubes that are at least half full. The information that smaller sample volumes can result in negative bias is presented in the package insert.

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):
None

4. Clinical cut-off:
None

5. Expected values/Reference range:
Reference intervals were determined for the VITROS HCY assay following NCCLS Guideline C28-A2. The reference interval is based on the central 95 percent of homocysteine values from an Ortho Clinical Diagnostics, Inc. study of 106 healthy female and 99 healthy male adults. Testing was performed on three VITROS 5,1 FS Chemistry System analyzers using 3 lots of VITROS HCY reagents. The reference range for males is 6.6-14.8 μmol/L and for females is 4.7-12.6 μmol/L.

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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