Cholesterol

K150654 · Randox Laboratories, Ltd. · CHH · Sep 29, 2015 · Clinical Chemistry

Device Facts

• Record ID: K150654
• Device Name: Cholesterol
• Applicant: Randox Laboratories, Ltd.
• Product Code: CHH · Clinical Chemistry
• Decision Date: Sep 29, 2015
• Decision: SESE
• Submission Type: Traditional
• Regulation: 21 CFR 862.1175
• Device Class: Class 1

Indications for Use

For the quantitative in vitro determination of Cholesterol in serum and plasma. Cholesterol measurements are used in the diagnosis and treatments of disorders involving excess cholesterol in the blood and lipid and lipoprotein metabolism disorders.

Device Story

Randox Cholesterol test system is a quantitative colorimetric assay for serum and plasma; utilizes RX Daytona Plus analyzer. Reagent (R1) contains cholesterol esterase, cholesterol oxidase, peroxidase, 4-aminoantipyrine, and phenol. Cholesterol esters hydrolyzed to free cholesterol; oxidized by cholesterol oxidase to produce hydrogen peroxide; reacts with phenol and 4-aminoantipyrine via peroxidase to form red quinoneimine complex. Absorbance measured at 510 nm/700 nm; intensity proportional to cholesterol concentration. Used in clinical laboratory settings by trained personnel. Output provides cholesterol concentration (mg/dL) to assist clinicians in diagnosing and treating lipid/lipoprotein metabolism disorders and atherosclerosis. Benefits include standardized monitoring of blood cholesterol levels.

Clinical Evidence

Bench testing only. Precision evaluated per CLSI EP5-A2 (CVs < 3%). Linearity per CLSI EP6-A (range 25-618 mg/dl). LoD 6.31 mg/dl; LoQ 23.2 mg/dl. Analytical specificity confirmed for hemoglobin, bilirubin, Intralipid, and ascorbic acid. Method comparison against predicate (n=107) yielded y=1.00x-4.77 (r=0.997). Matrix comparison for Li-Heparin (n=54) and K2-EDTA (n=50) plasma showed high correlation (r≥0.997) to serum.

Technological Characteristics

Quantitative colorimetric enzymatic assay. Reagents: 4-aminoantipyrine, phenol, peroxidase, cholesterol esterase, cholesterol oxidase, sodium azide. Measurement: absorbance at 510/700 nm. Form factor: liquid ready-to-use reagent. Connectivity: integrated with RX Daytona Plus analyzer. Standards: CLSI EP5-A2, EP17-A2, EP9-A2, EP6-A.

Indications for Use

Indicated for the quantitative in vitro determination of cholesterol in serum and plasma for the diagnosis and treatment of disorders involving excess blood cholesterol and lipid/lipoprotein metabolism disorders.

Regulatory Classification

Identification

A cholesterol (total) test system is a device intended to measure cholesterol in plasma and serum. Cholesterol measurements are used in the diagnosis and treatment of disorders involving excess cholesterol in the blood and lipid and lipoprotein metabolism disorders.

Predicate Devices

• Randox Laboratories Ltd, Cholesterol reagent (K923504)

Reference Devices

• Randox Assayed Multisera Level 2 (K942458)
• Randox Assayed Multisera Level 3 (K942458)
• Randox Calibration Serum Level 3 (K053153)

Submission Summary (Full Text)

{0}

1

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

A. 510(k) Number:
k150654

B. Purpose for Submission:
New submission

C. Measurand:
Total Cholesterol

D. Type of Test:
Quantitative colorimetric assay

E. Applicant:
Randox Laboratories Limited

F. Proprietary and Established Names:
Cholesterol

G. Regulatory Information:

| Product Code | Classification | Regulation Section | Panel |
| --- | --- | --- | --- |
| CHH | Class I, meets the limitation of exemption 21 CFR §862.9(c)(4) | 21 CFR §862.1175 Cholesterol (total) Test System | Chemistry (75) |

H. Intended Use:

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

2. Indication(s) for use:
For the quantitative in vitro determination of Cholesterol in serum and plasma.
Cholesterol measurements are used in the diagnosis and treatments of disorders involving excess cholesterol in the blood and lipid and lipoprotein metabolism disorders.

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

{1}

4. Special instrument requirements:

For use with the RX Daytona Plus analyzer (k131554)

I. Device Description:

The Randox Cholesterol test system is a one reagent system. The reagent (R1) is supplied in liquid ready-to-use form and contains 4-aminoantipyrine (0.23 mmol/L), Phenol 6.0 mmol/L, Peroxidase (≥ 0.50 U/ml), cholesterol esterase (≥0.20 U/mL) cholesterol oxidase (≥0.10 U/mL) and Sodium Azide (0.09%).

Materials required but not provided within the assay kit: Randox Assayed Multisera Level 2 and 3 (k942458), Randox Calibration Serum Level 3 (k053153) and RX series Saline.

J. Substantial Equivalence Information:

1. Predicate device name(s):

Randox Laboratories Ltd, Cholesterol enzymatic endpoint

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

k923504

3. Comparison with predicate:

| Similarities and Differences | | |
| --- | --- | --- |
| Item | Proposed Device | Predicate device (k923504) |
| Intended Use | The Cholesterol test system is a device intended for the quantitative in vitro determination of Cholesterol in serum and plasma. Cholesterol measurements are used in the diagnosis and treatment of lipid lipoprotein metabolism disorders and atherosclerosis | Same |
| Sample Type | Serum and Plasma (Li Heparin and K2-EDTA) | Same |
| Calibration Frequency | Every 28 days, with a change of reagent lot or as indicated by quality control procedures | Same |
| Measuring Range | 25 – 618mg/dL | 0.55 – 750mg/dL |

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

- CLSI EP5-A2: Evaluation of Precision Performance of Quantitative Measurement Methods; Approved Guideline -Second Edition
- CLSI EP17-A2: Evaluation of Detection Capability for Clinical Laboratory

{2}

Measurement Procedures; Approved Guideline -Second Edition

- CLSI EP9-A2: Method Comparison and Bias Estimation Using Patient Samples; Approved Guideline - Second Edition (Interim Revision)
- CLSI EP6-A: Evaluation of the Linearity of Quantitative Measurement Procedures: A Statistical Approach; Approved Guideline

## L. Test Principle:

Cholesterol esters in serum are completely hydrolyzed by cholesterol esterase to free cholesterol and free fatty acids. The cholesterol liberated by the esterase, plus any endogenous free cholesterol, are oxidized by cholesterol oxidase to yield hydrogen peroxide (H₂O₂), which then reacts oxidatively with phenol and 4-aminoantipyrine (AAP) in a reaction catalyzed by peroxidase, producing a red colored quinoneimine complex. The amount of colored complex formed, determined by quantitatively measuring the increase in absorbance at 510 nm/700 nm, is directly proportional to the cholesterol concentration in the sample.

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

### 1. Analytical performance:

#### a. Precision/Reproducibility:

Precision of the Cholesterol test system was evaluated according to CLSI EP5-A2. Two reagent lots and 2 RX Daytona Plus analyzers were utilized in the study. A native patient serum pool, altered serum sample pools, control pools and a calibrator serum sample were all evaluated. Two replicates of the serum samples were tested on two separate runs per day for 20 days, leading to the generation of 80 data points for each sample. The results of one representative lot are shown below:

| Sample | Mean (mg/dL) | Within-run | | Total | |
| --- | --- | --- | --- | --- | --- |
| | | SD | %CV | SD | %CV |
| Native Patient Pool | 32.4 | 1.17 | 3.6 | 3.35 | 10.3 |
| Serum Pool 1 | 177 | 3.56 | 2.0 | 4.82 | 2.7 |
| Control Pool 1 | 192 | 3.33 | 1.7 | 4.68 | 2.4 |
| Serum Pool 2 | 228 | 4.04 | 1.8 | 6.20 | 2.7 |
| Serum Pool 3 | 272 | 3.84 | 1.4 | 7.35 | 2.7 |
| Calibrator Serum | 285 | 4.22 | 1.5 | 5.59 | 2.0 |
| Control Pool 2 | 310 | 5.52 | 1.8 | 7.22 | 2.3 |
| Serum Pool 4 | 592 | 6.76 | 1.1 | 11.3 | 1.9 |

#### b. Linearity/assay reportable range:

A linearity study was performed following CLSI EP6-A by evaluating a dilution

{3}

series of serum samples containing the cholesterol analyte. Samples were prepared by mixing a high spiked serum sample with a low diluted serum sample to obtain 11 samples with concentrations spanning the claimed measuring range, 25 to 618 mg/dL. Each level was analyzed in replicates of five using two lots of Randox Cholesterol reagent on one RX Daytona Plus analyzer system. The results of the linear regression analysis of one representative lot are shown below:

$$
y = 1.00x - 5.49; r = 0.999
$$

The RX Daytona Plus analyzer is capable of auto diluting with a 1:15 dilution factor when measuring samples &gt; 619 mg/dL, and an auto-dilution study was conducted against a manual dilution and all results were within a pre-specified acceptance criteria.

The reportable range of the assay is 25 - 618 mg/dL.

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

**Traceability:**

The controls and calibrator to be used with this assay are manufactured by Randox Laboratories. Calibrator (Randox Calibration Serum Level 3) and controls (Randox Assayed Multisera Level 2 and Level 3) were previously cleared in k053153 and k942458 respectively. Randox Calibration Serum Level 3 is traceable to Cholesterol reference material NIST 1952a.

**Stability Studies:**

Stability of the Cholesterol kit is based on real-time stability study data of three kit lots. The stability study protocol and acceptance criteria was reviewed and found to be acceptable. The real time stability study supports a shelf life stability of 24 months when materials are stored at 2-8°C. Once opened, the kit is stable for 28 days at 2-8°C.

d. Detection limit:

A detection limit study was performed according to CLSI EP17-A2 guideline. Limit of Blank (LoB) was determined by running 4 blank samples (serum based matrix free of analyte) in 20 replicates across 3 days giving 60 determinations in total on the RX Daytona Plus analyzer, and LoB was the value at the 95th percentile. The Limit of Detection (LoD) was determined by evaluating 4 diluted patient serum sample pools in 20 replicates across 3 days for a total of 60 data points on a RX Daytona Plus analyzer. Limit of Quantitation (LoQ) was determined by running 4 diluted patient serum sample pools in 12 replicates across 5 days yielding a total of 60 results. Sponsor defines the LoQ as the lowest analyte concentration where the %CV is ≤ 20%.

| Analyte | LoB | LoD | LoQ |
| --- | --- | --- | --- |
| Cholesterol | 3.1 mg/dL | 6.31 mg/dL | 23.2 mg/dL |

The claimed measuring range is 25 - 619 mg/dL for the Randox Cholesterol Assay.

e. Analytical specificity:

{4}

Interference testing was performed in accordance with CLSI EP-7A2 with 2 different concentrations of cholesterol (150 mg/dL and 250 mg/dL) using one RX Daytona Plus analyzer and one reagent lot. Samples with added potential interferents were tested in replicates of 10, and the mean recoveries were compared to samples without interferent. The sponsor defined no significant interference as ≤10 % difference from the control sample.

Summary of interference study showing highest concentration that does not interfere:

| Interferent | Concentration at which no interference was observed |
| --- | --- |
| Hemoglobin | 750 mg/dL |
| Total Bilirubin | 60 mg/dL |
| Bilirubin (conjugated) | 60 mg/dL |
| Intralipid | 1000 mg/dL |
| Ascorbic acid | 6 mg/dL |

f. Assay cut-off:

Not applicable.

2. Comparison studies:

a. Method comparison with predicate device:

A method comparison study was performed in accordance with CLSI EP9-A2. A total of 107 serum patient samples spanning the range of 25 to 599.5 mg/dL were analyzed with the candidate cholesterol assay on the Randox Daytona Plus system versus the predicate device. Of the 107 samples, 9 samples were diluted or spiked. All samples were analyzed in singlicate. Results of the linear regression are presented below:

| Slope (95% CI) | Intercept (95% CI) | R |
| --- | --- | --- |
| 1.00 (0.98 to 1.01) | -4.77 (-7.99 to -1.57) | 0.997 |

b. Matrix comparison:

A matrix comparison study was conducted using matched sets of serum and plasma (lithium-heparin and K₂EDTA plasma) samples which spanned the reportable range of the assay. The samples (6 diluted and 4 spiked) were analyzed on the RX Daytona Plus analyzer. The details and results of the study using simple linear regression analysis are as follows:

| Sample Type | n | Intercept | Slope (95% CI) | R | Sample range tested |
| --- | --- | --- | --- | --- | --- |

{5}

| Serum vs. lithium Heparin | 54 | -6.54 (-12.01 to -1.07) | 1.01 (0.99 to 1.03) | 0.997 | 25 to 613 mg/dL |
| --- | --- | --- | --- | --- | --- |
| Serum versus K2-EDTA plasma | 51 | 2.73 (-1.39 to 6.86) | 0.99 (0.97 to 1.00) | 0.998 | 29 to 603 mg/dL |

Based on the study data, the sponsor claims that serum, lithium heparin and $\mathbf{K}^2$ -EDTA plasma samples are acceptable for use with the Randox cholesterol 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):

Not applicable.

# 4. Clinical cut-off:

Not applicable.

# 5. Expected values/Reference range:

$&lt; 200 \mathrm{mg} / \mathrm{dL}$ - Desirable blood cholesterol

200-239- Borderline High Blood Cholesterol

$\geq 240 \mathrm{mg} / \mathrm{dL}$ -High Blood Cholesterol

$^{1}$ Third Report of the National Cholesterol Education Program (NCEP) Expert Panel on Detection, Evaluation and treatment of High Blood Cholesterol in Adults (Adult Treatment Panel III). JAMA Publication, Vol. 285, No. 19, P2486-2497; 2001.

# 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.