CATACHEM, INC., BILE ACIDS REAGENTS AND/OR BILE ACIDS TEST KIT

K062503 · Catachem, Inc. · KWW · Jan 19, 2007 · Clinical Chemistry

Device Facts

• Record ID: K062503
• Device Name: CATACHEM, INC., BILE ACIDS REAGENTS AND/OR BILE ACIDS TEST KIT
• Applicant: Catachem, Inc.
• Product Code: KWW · Clinical Chemistry
• Decision Date: Jan 19, 2007
• Decision: SESE
• Submission Type: Traditional
• Regulation: 21 CFR 862.1177
• Device Class: Class 2

Indications for Use

Catachem, Inc. Bile Acids assay is an In-Vitro diagnostic enzymatic assay for the quantitative determination of total bile acids in human serum. Measurement of bile acids in human serum, and increased reported levels are representative of specific liver disease. For In-Vitro diagnostic use only Bile Acids Calibrator. (For In-Vitro diagnostic use only) The bile acids assay contains a calibrator designed for the calibration of the Bile Acids method. Bile Acids Controls (For In-Vitro diagnostic use only) The bile acids assay also contains control Level-I and control Level-II. These controls are designed to monitor the performance of the Bile Acids assay.

Device Story

In-vitro enzymatic assay for quantitative determination of total bile acids in human serum; utilizes COBAS MIRA analyzer. Principle: 3α-hydroxysteroid dehydrogenase oxidizes bile acids to 3-keto hydroxyl bile acids, reducing NAD+ to NADH; diaphorase then oxidizes NADH to NAD+, reducing nitrotetrazolium blue (NBT) to a formazan dye. Color intensity at 540 nm is proportional to bile acid concentration. Used in clinical laboratory settings by trained personnel. Output provides numerical bile acid concentration (μmol/L) to assist clinicians in diagnosing or monitoring liver disease. Includes liquid reagents, calibrator, and controls.

Clinical Evidence

No clinical data provided; substantial equivalence based on bench testing and performance characteristics of the enzymatic assay.

Technological Characteristics

Enzymatic colorimetric assay. Reagents: 3-α-hydroxysteroid dehydrogenase, diaphorase, NAD+, nitrotetrazolium blue. Form factor: Liquid, ready-to-use reagents. Energy source: Spectrophotometric measurement (540 nm) via automated analyzer (COBAS MIRA). Calibration: Multi-point using bovine serum-based calibrator. Controls: Bovine serum-based Level-I and Level-II. Standards: CLSI EP05-A2, EP06-A, EP07-A, EP09-A2, EP17-A.

Indications for Use

Indicated for the quantitative determination of total bile acids in human serum to aid in the diagnosis of liver disease. For prescription use only.

Regulatory Classification

Identification

A cholylglycine test system is a device intended to measure the bile acid cholylglycine in serum. Measurements obtained by this device are used in the diagnosis and treatment of liver disorders, such as cirrhosis or obstructive liver disease.

Submission Summary (Full Text)

{0}

510(K) SUBSTANTIAL EQUIVALENCE DETERMINATION DECISION SUMMARY ASSAY ONLY TEMPLATE

A. 510(k) Number:
k062503

B. Purpose for Submission
New device

C. Measurand:
Bile acid cholylglycine

D. Type of Test:
Enzymatic Colorimetric Assay

E. Applicant:
Catachem, Inc.

F. Proprietary and Established Names:
Bile Acids (Liquid) Reagents
Bile Acids Calibrator
Bile Acids Controls

G. Regulatory Information:
1. Regulation Section
21 CFR §862.1177, Cholylglycine test system
21 CFR §862.1150, Calibrator
21 CFR §862.1660, Quality Control Material
2. Classification:
Class II (Reagent, Calibrator)
Class I Reserved (Control)
3. Product Code:
KWW; JIS; JJX
4. Panel
Clinical Chemistry (75)

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

2. Indication(s) for use:
Catachem, Inc. Bile Acids assay is an In-Vitro diagnostic enzymatic assay for the quantitative determination of total bile acids in human serum. Measurement of bile acids in human serum, and increased reported levels are representative of specific liver disease.

Bile Acids Calibrator
The bile acids assay contains a calibrator designed for the calibration of the Bile Acids method.

1

{1}

2

# Bile Acids Controls

The Bile Acids assay also contains Control Level-I and Control Level-II. These controls are designed to monitor the performance of the Bile Acids assay.

3. Special conditions for use statement(s):

For Prescription Use only.

4. Special instrument requirements:

COBAS MIRA analyzer

# I. Device Description:

The DiscretPak Liquid Bile Acids consists of:

1. Two reagents - Enzyme Color Reagent containing buffer, pH 7.8, Nitrotetrazolium blue 0.6 mmol/L, 3-α-hydroxysteroid dehydrogenase ≥800 units/L, Diaphorase ≥5000 units/L and preservatives and stabilizer. The Bile Acids Activator Reagent is an aqueous solution, pH ≤ 3.5 with preservative and stabilizer.
2. Calibrator and Control Level-I and Control Level-II are bovine serum based and contain pure bile acid with preservative and stabilizer.

# J. SUBSTANTIAL EQUIVALENCE INFORMATION

1. Predicate device name(s):

Bile Acids Reagents

2. Predicate 510(k) number(s):

k872296

3. Comparison with predicate:

4.

| Similarities | | |
| --- | --- | --- |
| Item | Device | Predicate |
| Intended Use | For the quantitative enzymatic determination of bile acids in serum. | For the quantitative enzymatic determination of bile acids in serum. |
| Method Principle | 1. Bile acids are oxidized to 3-Keto hydroxyl bile acids by the enzyme 3-α-hydroxysteroid dehydrogenase with reduction of NAD to NADH.
2. NADH is oxidized to NAD by the action of the enzyme diaphorase where NBT dye is reduced to form a formazan dye with maximum absorbance at 540 nm. The intensity of the color produced is proportional to the concentration of the bile acids in the serum sample. | 1. Bile acids are oxidized to 3-Keto hydroxyl bile acids by the enzyme 3-α-hydroxysteroid dehydrogenase with reduction of NAD to NADH.
2. NADH is oxidized to NAD by the action of the enzyme diaphorase where NBT dye is reduced to form a formazan dye with maximum absorbance at 540 nm. The intensity of the color produced is proportional to the concentration of the bile acids in the serum sample. |
| Applications | Manual and automated. | Manual and automated. |
| Calibration | Bile acids concentration in the serum sample is calculated using a calibrator. | Bile acids concentration in the serum sample is calculated using a calibrator. |

{2}

| Similarities | | |
| --- | --- | --- |
| Item | Device | Predicate |
| Quality Control | The reliability of test results is monitored by routine use of bile acids control sera. | The reliability of test results is monitored by routine use of bile acids control sera. |
| Expected Values | 0-8.0 μmol/L | 0-8.0 μmol/L |
| Analytical Range | 1.0-200 μmol/L | 1.9-200 μmol/L |
| Linear Range | Bile acids method linear to 200 μmol/L | Bile acids method linear to 200 μmol/L |
| Differences | | |
| --- | --- | --- |
| Item | Device | Predicate |
| Reagents | Two liquid reagents, ready for use. | Two lyophilized reagents, requires preparation |
| Working Reagent Stability | 60 days at 2-8 °C | One week at 2-8 °C |
| Manual Method Serum Sample Dilution | 0.05 ml serum sample plus 0.5 ml reagent for a total sample dilution of 1:11 | 0.2 ml serum sample plus 0.5 ml reagent for a total sample dilution of 1:3.5 |
| Method Sensitivity | 0.000958 O.D. / μmol/L, automated-method, COBAS MIRA analyzer, 1:13 sample dilution. | 0.000665 O.D. / μmol/L, automated-method, COBAS MIRA analyzer, 1:13 sample dilution. |
| Interference Substances | Ascorbic acid ≤50 mg/dl Bilirubin ≤1.7 mg/dl Hemoglobin ≤300 mg/dl Lipemia (triglycerides) ≤1000 mg/dl | Ascorbic acid Not reported Bilirubin Not reported Hemoglobin Not reported Triglycerides Not reported |

# K. STANDARD/GUIDANCE DOCUMENT REFERENCED (IF APPLICABLE)

CLSI EP05-A2: Evaluation of Precision Performance of Clinical Chemistry Devices; Approved Guideline-Second Edition.

CLSI EP06-A: Evaluation of the linearity of Quantitative Analytical Methods; Approved Guideline.

CLSI EP07-A: Interference Testing in Clinical Chemistry; Proposed Guideline.

CLSI EP09-A2: Method Comparison and bias Estimation Using Patient Samples; Approved Guideline-Second Edition.

CLSI EP17-A: Protocol for Determination of Limits of Detection and Limits of Quantitation; Approved Guideline

# L. Test Principle

In this Bile Acids procedure, $3\alpha$ -hydroxy Bile Acids are converted to corresponding 3-keto hydroxy Bile Acids by the action of the enzyme $3\alpha$ -hydroxysteroid dehydrogenase $(3\alpha$ -HSDH) with concomitant reduction of $\mathrm{NAD+}$ to NADH. The NADH thus produced is subsequently oxidized to $\mathrm{NAD+}$ in a diaphorase-catalyzed reaction where nitrotetrazolium

{3}

blue (NBT) is reduced to form a formazan dye, which has an absorption maximum at 540 nm. The intensity of the color produced is directly proportional to the concentration of Bile Acids in the serum sample.

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

### 1. Analytical Performance:

a. Precision /Reproducibility:

Serum samples prepared in-house were spiked with bile acids to obtain four different concentrations in $\mu \mathrm{mol} / \mathrm{L}$. These samples were assayed on the analyzer COBAS MIRA PLUS for a period of seven days 3 times per day. Results were tabulated and the statistics were worked out to arrive at the results claimed in the device.

| SAMPLE TYPE | BILE ACIDS | WITHIN-RUN | | DAY-DAY | |
| --- | --- | --- | --- | --- | --- |
| | MEAN | SD | CV | SD | CV |
| | μmol/L | μmol/L | % | μmol/L | % |
| SERUM | 6.0 | 0.23 | 3.85 | 0.26 | 4.51 |
| SERUM | 25 | 0.77 | 3.0 | 1.23 | 5.01 |
| SERUM | 150 | 2.84 | 1.86 | 7.30 | 5.08 |

b. Linearity/ Assay reportable range.

The measuring range of this device is $1 - 200\ \mu \mathrm{mol} / \mathrm{L}$. Linearity was evaluated following the recommendations of CLSI EP6-A on an automated analyzer.

Two serum samples were prepared with $99\%$ pure bile acids (cholic acid) to obtain a lower concentration and a high concentration. Seven additional serum samples were prepared by combining the low $(2.1\mu \mathrm{mol} / \mathrm{L})$ and the high $(228.5\mu \mathrm{mol} / \mathrm{L})$ serum samples. A total of 9 serum dilutions and concentrations spanning the desired measuring range $(2 - 200\mu \mathrm{mol} / \mathrm{L})$ were assayed in random order in replicates of four. The resulting linear regression statistics were: Observed = 1.0037(Expected) - 1.6699.

| Device Bile Acids Linearity-Serum | |
| --- | --- |
| EXPECTED VALUES | OBSERVED VALUES |
| 2.1 | 2.10 |
| 30.1 | 29.80 |
| 58.7 | 55.55 |
| 87.0 | 85.15 |
| 115.3 | 112.70 |
| 146.6 | 143.20 |
| 171.9 | 171.40 |
| 200.2 | 200.85 |
| 228.5 | 228.50 |

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

Projected and real life stability studies of the bile acid reagents were conducted. Accelerated stability testing of the bile acids reagents were done at $30^{\circ}\mathrm{C}$ and $37^{\circ}\mathrm{C}$ to project the shelf life of reagents. Real life stability tests are in progress at $2 - 8^{\circ}\mathrm{C}$. Based on

{4}

the accelerated experiments the sponsor projects reagents to be stable for 12 months. These stability experiments were done using reagent formulations packaged ready for use by the consumer. Specific concentrations of the essential components in the reagent formulation are disclosed in the bile acids package insert.

The device bile acids require a Calibrator to compute the bile acids concentration of the unknown serum samples. The device bile acids also require a Control Level-I and Level-II to monitor the quality of the assay. These three required components are value-assigned by performing multiple determinations using an automated analyzer. The Calibrator and Control Level I and Level II are traceable to an in-house reference master lot which was cross referenced with the predicate bile acids assay calibrator and assessed for agreement. The device bile acids calibrator has a nominal value of $100\mu \mathrm{mol} / \mathrm{L}$ and the controls level I and II have nominal values of 50 and $150\mu \mathrm{mol} / \mathrm{L}$ respectively.

# d. Detection Limit

Limits of Quantitation (LoQ) and Limits of Detection (LoD)

The lower limit of detection and quantitation was established following the guidelines of CLSI (Formerly NCCLS) EP17-A. The criterion for limits of quantitation is the concentration of the analyte which has imprecision $\leq 20\%$ cv.

The lower limit of sensitivity of the device bile acids was determined based on the LoQ. To establish the LoQ, serum bile acids assays were done in between zero-baseline and the established linear low (2 $\mu$ mol/L). Three runs were conducted on an automated analyzer, using replicates of 20 in each case. The assays revealed a LoQ value of $1.7\mu \mathrm{mol} / \mathrm{L}$ . The LoD was determined to be $0.9\mu \mathrm{mol} / \mathrm{L}$ .

| Device Bile Acids Limits of Quantitation and Detection-Serum Samples | |
| --- | --- |
| LoD Results | LoQ Results |
| 0.001 OD, 0.9 μmol/L | 1.7 μmol/L, % CV 20.0 |

# e. Analytical Specificity

Interference studies were conducted following the guidelines of CLSI EP07-A.

Potential substances which may affect the accuracy of the device were evaluated using serum samples. The study included ascorbic acid, bilirubin, hemoglobin and lipemia (triglycerides). The level of interference was considered acceptable if there was no more than $\pm 10\%$ difference between the iterferant result and the reference result.

| Interfering Substance | Concentration mg/dL |
| --- | --- |
| Ascorbic Acid | ≤ 50 mg/dL |
| Bilirubin | ≤ 1.7 mg/dL |
| Hemoglobin | ≤ 300 mg/dL |
| Lipemia(Triglycerides) | ≤ 1000 mg/dL |

f. Assay Cut-off

{5}

Not applicable

## 2. Comparison Studies:

a. Method comparison with predicate device:

Method comparison studies were performed following the guidelines of CLSI EP09-A2 on an automated analyzer.

A correlation of the device bile acids with the predicate was conducted. Serum samples (n=36) obtained from a local hospital and 30 spiked serum samples (>60 μmol/L) prepared in-house were used in the comparison study. The total number of samples was 66 and the observed analytical range was 1.0-159.8 μmol/L.


## BILE ACIDS COMPARISON REGRESSION ANALYSIS

N = 66
Range = 1.0-159.8 μmol/L.
Mean of X = 50.95 μmol/L.
Mean of Y = 48.50 μmol/L.
Y = 0.924X + 4.3
R = 0.9958
Sy.x = 4.079

b. Matrix comparison

Not applicable.

## 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)

Not applicable

## 4. Clinical cut-off:

Not applicable

## 5. Expected Values/ Reference Ranges:

Expected Values 0.0-8.0 μmoles/L

(1. Mashige F, Osuga T, Tanka N, Yamanaka M: Continuous -flow determination of bile acids in serum and its clinical application. Clin Chem 24: 1150, 1978. & 2. Mashige F, Tanaka N, Maki A, Kamei S, Yamanaka M: Direct spectrophotometry of total bile acids in serum, Clin Chem 27:1352, 1981.)

{6}

N. Proposed Labeling:
The labeling is sufficient to and 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.

7