LDLC3 LDL-Cholesterol Gen.3

K143691 · Roche Diagnostics Operations (Rdo) · LBR · Jan 28, 2015 · Clinical Chemistry

Device Facts

• Record ID: K143691
• Device Name: LDLC3 LDL-Cholesterol Gen.3
• Applicant: Roche Diagnostics Operations (Rdo)
• Product Code: LBR · Clinical Chemistry
• Decision Date: Jan 28, 2015
• Decision: SESE
• Submission Type: Traditional
• Regulation: 21 CFR 862.1475
• Device Class: Class 1

Intended Use

The LDL-Cholesterol Gen. 3 assay is intended for use as an in vitro test for the quantitative determination of LDL-Cholesterol in human serum and plasma on Roche/Hitachi cobas c systems.

Device Story

LDLC3 LDL-Cholesterol Gen. 3 is a homogeneous enzyme colorimetric assay for quantitative measurement of LDL-cholesterol in human serum and plasma. Used on Roche/Hitachi cobas c clinical chemistry analyzers; operated by laboratory professionals. Principle: surfactants selectively solubilize LDL; cholesterol esterase and oxidase break down LDL-cholesterol; peroxidase reacts with 4-aminoantipyrine and EMSE to produce a red-purple dye. Color intensity, measured photometrically, is directly proportional to cholesterol concentration. Output provides quantitative LDL-cholesterol levels to assist clinicians in diagnosing and managing lipid disorders, atherosclerosis, and liver/renal diseases.

Clinical Evidence

Bench testing only. Performance validated per CLSI guidelines: LoB (0.406 mg/dL), LoD (0.99 mg/dL), LoQ (2.28 mg/dL). Precision (CLSI EP5-A2) showed CVs 0.7-2.5%. Linearity (CLSI EP6-A) confirmed 3.87-549 mg/dL range. Method comparison (n=100) against predicate yielded Passing/Bablok regression y = 0.984x + 0.786, r = 0.999. Interference testing confirmed no significant impact from common drugs, VLDL, HDL, chylomicrons, or endogenous substances (hemoglobin, lipemia, bilirubin) within specified limits.

Technological Characteristics

Homogeneous enzyme colorimetric assay. Reagents: R1 (Bis-tris buffer, ascorbic oxidase, peroxidase, BSA) and R2 (MOPS buffer, EMSE, cholesterol esterase, cholesterol oxidase, peroxidase, BSA). Sensing: photometric measurement of red-purple dye intensity. Connectivity: designed for Roche/Hitachi cobas c systems. Software: uses Roche-validated biometric regression analysis tool for linearity verification.

Indications for Use

Indicated for quantitative determination of LDL-cholesterol in human serum and plasma for the diagnosis and treatment of lipid disorders (e.g., diabetes mellitus), atherosclerosis, and various liver and renal diseases.

Regulatory Classification

Identification

A lipoprotein test system is a device intended to measure lipoprotein in serum and plasma. Lipoprotein measurements are used in the diagnosis and treatment of lipid disorders (such as diabetes mellitus), atherosclerosis, and various liver and renal diseases.

Submission Summary (Full Text)

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Food and Drug Administration 10903 New Hampshire Avenue Document Control Center - WO66-G609 Silver Spring, MD 20993-0002

ROCHE DIAGNOSTICS OPERATIONS (RDO) NOEL MENCIAS REGULATORY AFFAIRS CONSULTANT 9115 HAGUE ROAD INDIANAPOLIS IN 46250

January 28, 2015

Re: K143691

Trade/Device Name: LDLC3 LDL-Cholesterol Gen.3 Regulation Number: 21 CFR 862.1475 Regulation Name: Lipoprotein test system Regulatory Class: I, Meets limitations of the exemption as per 21 CFR 862.9 (c)(4) Product Code: LBR Dated: December 23, 2014 Received: December 24, 2014

Dear Noel Mencias:

We have reviewed your Section 510(k) premarket notification of intent to market the device referenced above and have determined the device is substantially equivalent (for the indications for use stated in the enclosure) to legally marketed predicate devices marketed in interstate commerce prior to May 28, 1976, the enactment date of the Medical Device Amendments, or to devices that have been reclassified in accordance with the provisions of the Federal Food, Drug, and Cosmetic Act (Act) that do not require approval of a premarket approval application (PMA). You may, therefore, market the device, subject to the general controls provisions of the Act. The general controls provisions of the Act include requirements for annual registration, listing of devices, good manufacturing practice, labeling, and prohibitions against misbranding and adulteration. Please note: CDRH does not evaluate information related to contract liability warranties. We remind you, however, that device labeling must be truthful and not misleading.

If your device is classified (see above) into either class II (Special Controls) or class III (PMA), it may be subject to additional controls. Existing major regulations affecting your device can be found in the Code of Federal Regulations, Title 21, Parts 800 to 898. In addition, FDA may publish further announcements concerning your device in the Federal Register.

Please be advised that FDA's issuance of a substantial equivalence determination does not mean that FDA has made a determination that your device complies with other requirements of the Act or any Federal statutes and regulations administered by other Federal agencies. You must comply with all the Act's requirements, including, but not limited to: registration and listing (21 CFR Part 807); labeling (21 CFR Parts 801 and 809); medical device reporting (reporting of medical device-related adverse events) (21 CFR 803); good manufacturing practice requirements as set forth in the quality systems (QS) regulation (21 CFR Part 820); and if applicable, the electronic product radiation control provisions (Sections 531-542 of the Act); 21 CFR 1000-1050.

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If you desire specific advice for your device on our labeling regulations (21 CFR Parts 801 and 809), please contact the Division of Industry and Consumer Education at its toll-free number [phone] or [phone] or at its Internet address

http://www.fda.gov/MedicalDevices/ResourcesforYou/Industry/default.htm. Also, please note the regulation entitled, "Misbranding by reference to premarket notification" (21 CFR Part 807.97). For questions regarding the reporting of adverse events under the MDR regulation (21 CFR Part 803), please go to

http://www.fda.gov/MedicalDevices/Safety/ReportaProblem/default.htm for the CDRH's Office of Surveillance and Biometrics/Division of Postmarket Surveillance.

You may obtain other general information on your responsibilities under the Act from the Division of Industry and Consumer Education at its toll-free number [phone] or [phone] or at its Internet address

http://www.fda.gov/MedicalDevices/ResourcesforYou/Industry/default.htm.

Sincerely yours,

# Katherine Serrano -S

For : Courtney H. Lias, Ph.D. Director Division of Chemistry and Toxicology Devices Office of In Vitro Diagnostics and Radiological Health Center for Devices and Radiological Health

Enclosure

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#### Indications for Use

510(k) Number (if known) k143691

Device Name LDLC3 LDL-Cholesterol Gen.3

#### Indications for Use (Describe)

The LDL-Cholesterol Gen. 3 assay is an in-vitro test for the quantitative determination of LDL-cholesterol in human serum and plasma on Roche/Hitachi cobas c systems. Lipoprotein measurements are used in the diagnosis and treatment of lipid disorders (such as diabetes mellitus), atherosclerosis, and various liver and renal diseases.

| Type of Use (Select one or both, as applicable) | |
|--------------------------------------------------|---------------------------------------------|
| × Prescription Use (Part 21 CFR 801 Subpart D) | Over-The-Counter Use (21 CFR 801 Subpart C) |

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| Date prepared: | December 23, 2014 |
|------------------------|---------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------|
| Purpose of submission | In accordance with 21 CFR 807.87, Roche Diagnostics hereby submits official notification as required by Section 510(k) of the Federal Food, Drug and Cosmetics Act of our intention to market the device described in this Premarket Notification [510(k)]. This candidate device is a new reagent that was developed by Roche Diagnostics. The previous generation of reagent, LDL-Cholesterol plus 2nd generation, was cleared in 510(k) k974733 and serves as the predicate device. The candidate and predicate devices use the same calibrator and controls. Only the reagents differ. This submission presents data to support clearance of this new reagent. |
| Measurand | Lipoprotein |
| Type of test | Quantitative homogeneous enzyme colorimetric method |
| Applicant | Noel B. Mencias, Regulatory Affairs Consultant Roche Diagnostics 9115 South Hague Road Indianapolis, IN 46250 Telephone: [phone] Fax: [phone] Email: [e-mail] |
| Candidate device names | Proprietary name: LDLC3 LDL-Cholesterol Gen. 3 Common name: LDL-Cholesterol Gen. 3 Classification name: Lipoprotein Test System (21 CFR 862.1475) |

## 510(k) Summary for LDLC3 LDL-Cholesterol Gen. 3

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| Regulatory<br>information | Product Code | Classification | Regulation | Panel |
|----------------------------------------------------------------------------------------------------------------------|------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------|----------------|----------------------------------------------|-----------------------------|
| | LBR | Class I | 21 CFR 862.1475<br>(Lipoprotein test system) | Clinical<br>Chemistry<br>75 |
| LDLC3 LDL-Cholesterol Gen. 3 meets the limitation of exemption per 21<br>CFR 862.9 (c)(4) - for cardiovascular risk. | | | | |
| Intended use | The LDL-Cholesterol Gen. 3 assay is intended for use as an in vitro test for<br>the quantitative determination of LDL-Cholesterol in human serum and<br>plasma on Roche/Hitachi cobas c systems. | | | |
| Indications for<br>use | The LDL-Cholesterol Gen. 3 assay is an in-vitro test for the quantitative<br>determination of LDL-cholesterol in human serum and plasma on<br>Roche/Hitachi cobas c systems. Lipoprotein measurements are used in the<br>diagnosis and treatment of lipid disorders (such as diabetes mellitus),<br>atherosclerosis, and various liver and renal diseases. | | | |
| Special<br>conditions for<br>use | For prescription use only. | | | |
| Special<br>instrument<br>requirements | For use on the Roche/Hitachi cobas c clinical chemistry analyzer. | | | |
| | Continued on next page | | | |

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| Candidate<br>device<br>description | The LDL-Cholesterol Gen. 3 assay is a homogeneous enzyme colorimetric<br>assay which provides the quantitative measurement of LDL-cholesterol in<br>human serum and plasma.<br><br>Reagents are packaged in a cassette labeled with their instrument positioning<br>R1 (Reagent 1) and R2 (Reagent 2). |
|------------------------------------|------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------|
| | R1 contains Bis-trisb) buffer: 20.1 mmol/L, pH 7.0; 4-<br>aminoantipyrine:0.98 mmol/L; ascorbic oxidase (AOD, Acremonium<br>spec.): ≥ 66.7 µkat/L; peroxidase (recombinant from Basidiomycetes):<br>≥ 166.7 µkat/L; BSA: 4.0 g/L; preservative R2 contains MOPSC) buffer: 20.1 mmol/L, pH 7.0; EMSE: 2.16<br>mmol/L, cholesterol esterase (Pseudomonas spec.): ≥ 33.3 µkat/L;<br>cholesterol oxidase (recombinant from E.coli)): ≥ 31.7 µkat/L;<br>peroxidase (recombinant from Basidiomycetes): ≥ 333.3 µkat/L;<br>BSA: 4.0 g/L; detergents; preservative |
| Predicate<br>device | Roche Diagnostics claims substantial equivalence to LDL-Cholesterol plus<br>2nd generation reagent on the cobas c 501. The reagent was originally cleared<br>in k974733 on the Boehringer Mannheim/Hitachi clinical chemistry<br>analyzers, and later cleared in a Special 510(k) k012287 on COBAS<br>INTEGRA. The application to the cobas c 501 analyzer was cleared on<br>October 3, 2006 in k060373/A001 following the FDA Policy Document<br>"Replacement Reagent and Instrument Family Policy – 12/11/2003." |

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Substantial The following table compares the similar features of the candidate device to Equivalence – the predicate device that was cleared in 510(k) k974733. Assay Similarities

| Assay Comparison Similarities | | | |
|---------------------------------|------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------|-----------------------------------------------------------|--|
| Feature | Predicate Device:<br>LDL_C Cholesterol Plus 2nd<br>generation | Candidate Device:<br>LDLC3 LDL-Cholesterol Gen. 3 | |
| Intended Use | In vitro test for the quantitative<br>determination of LDL-Cholesterol in<br>human serum and plasma on<br>Roche/Hitachi cobas c systems. | Same | |
| Sample Types | Human Serum and Plasma | Same | |
| Test Principle | Homogenous enzymatic colorimetric<br>assav | Same | |
| Reagent Shelf Life<br>Stability | 2-8 ℃ until expiration date | Same | |
| Reagent On-Board<br>Stability | 12 weeks | Same | |
| Measuring Range | 0.10 - 14.2 mmol/L (3.86 - 548 mg/dL) | 3.87 -549 mg/dL | |
| Lower Limit of<br>Measurement | LDL(lower detection limit) = 0.10<br>mmol/L (3.866 mg/dL) | LoB = 0.406 mg/dL<br>LoD = 0.99 mg/dL<br>LoQ = 2.28 mg/dL | |
| Expected Values | Adult levels:<br>Optimal: < 2.59 mmol/L (< 100<br>mg/dL)<br>Near optimal/above optimal: 2.59-3.34<br>mmol/L (100-129 mg/dL)<br>Borderline high: 3.37-4.12 mmol/L<br>(130-159 mg/dL)<br>High 4.14-4.89 mmol/L (160-189<br>mg/dL)<br>Very high: ≥ 4.92 mmol/L (≥ 190<br>mg/dL) | Same | |
| Traceability | This method has been standardized<br>against the beta quantification method<br>as defined in the recommendations in<br>the LDL Cholesterol Method<br>Certification Protocol for Manufacturers | Same | |

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#### Substantial Equivalence – Assay Similarities (continued)

| Assay Comparison Similarities | | | |
|-------------------------------|------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------|------|--|
| Calibrator | Calibrator for automated systems<br>(C.f.a.s) Lipids and deionized water as<br>the zero calibrator. | Same | |
| | (C.f.a.s. cleared for use with LDL-<br>Cholesterol in k011658 | | |
| Calibration<br>frequency | Recalibrate after reagent lot change and<br>as required following quality control<br>procedures | Same | |
| Controls | Precinorm L<br>Precipath HDL/LDL-C<br>PreciControl ClinChem Multi 1<br>PreciControl ClinChem Multi 2<br>PreciControl ClinChem Multi 1 and<br>PreciControl ClinChem Multi 2 were<br>cleared for use with LDL C in 510(k)<br>k102016 | Same | |

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The following table compares the differences of the candidate device to the Substantial Equivalence – predicate device that was cleared in 510(k) k974733 Assay Difference

| Assay Comparison Differences | | |
|------------------------------|----------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------|-----------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------|
| Feature | Predicate Device:<br>LDL_C Cholesterol Plus 2nd<br>generation | Candidate Device:<br>LDLC3 LDL-Cholesterol Gen. 3 |
| Test Principle | In the presence of peroxidase, the<br>hydrogen peroxide generated<br>reacts with 4-aminoantipyrine and<br>HSDA to form a purple-blue dye.<br>The color intensity of this dye is<br>directly proportional to the<br>cholesterol concentration and is<br>measured photometrically. | In the presence of peroxidase, the<br>hydrogen peroxide generated reacts with<br>4-aminoantipyrine and EMSE to form a<br>red purple dye. The color intensity of<br>this dye is directly proportional to the<br>cholesterol concentration and is<br>measured photometrically. |
| Reagent Composition | R1 MOPS (3-morpholinopropane<br>sulfonic acid) buffer: 20.1 mmol/L,<br>pH 6.5; HSDA: 0.96 mmol/L;<br>ascorbate oxidase (Eupenicillium<br>spec., recombinant): ≥50 µkat/L;<br>peroxidase (horseradish): ≥167<br>µkat/L; preservative | R1 Bis-tris buffer: 20.1 mmol/L, pH<br>7.0; 4-aminoantipyrine:<br>0.98 mmol/L; ascorbic oxidase (AOD,<br>Acremonium spec.):<br>≥ 66.7 µkat/L; peroxidase (recombinant<br>from Basidiomycetes): ≥ 166.7 µkat/L;<br>BSA: 4.0 g/L; preservative |
| | R2 MOPS (3-morpholinopropane<br>sulfonic acid) buffer: 20.1 mmol/L,<br>pH 6.8; MgSO4·7H2O: 8.11<br>mmol/L; 4-aminoantipyrine: 2.46<br>mmol/L; cholesterol esterase<br>(Pseudomonas spec.): ≥50 µkat/L;<br>cholesterol oxidase (Brevibacterium<br>spec., recombinant): ≥33.3 µkat/L;<br>peroxidase (horseradish): ≥334<br>µkat/L; detergent; preservative | R2 MOPS buffer: 20.1 mmol/L, pH 7.0;<br>EMSE: 2.16 mmol/L,<br>cholesterol esterase (Pseudomonas<br>spec.): ≥ 33.3 µkat/L; cholesterol<br>oxidase (recombinant from E.coli)): ≥<br>31.7 µkat/L; peroxidase (recombinant<br>from Basidiomycetes): ≥ 333.3 µkat/L;<br>BSA: 4.0 g/L; detergents; preservative |

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| Summary of<br>tests | The following performance data were provided in support of the substantial<br>equivalence determination:<br>Limit of Blank according to CLSI EP17-A2<br>Limit of Detection according to CLSI EP17-A2<br>Limit of Quantitation according to CLSI EP17-A2<br>Precision according to CLSI EP5-A2<br>Linearity according to CLSI EP6-A<br>Rerun Function Check (Post Dilution Factor)<br>Recovery in Controls<br>Method Comparison<br>Verification of plasma as sample material<br>Drug Interferences<br>Endogenous Interferences in serum/plasma<br>Stability & Calibration frequency<br>All performance specifications were met. |
|---------------------|--------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------|
| Test Principle | Cholesterol esters and free cholesterol in LDL are measured on the basis of<br>a cholesterol enzymatic method using cholesterol esterase and cholesterol<br>oxidase in the presence of surfactants which selectively solubilizes only<br>LDL. The enzyme reactions to the lipoproteins other than LDL are inhibited<br>by surfactants and a sugar compound. Cholesterol in HDL, VLDL and<br>chylomicron is not determined. |
| | Cholesterol esters are broken down quantitatively into free cholesterol and<br>fatty acids by cholesterol esterase. |
| | In the presence of oxygen, cholesterol is oxidized by cholesterol oxidase to<br>Δ4-cholestenone and hydrogen peroxide. |
| | In the presence of peroxidase, the hydrogen peroxide generated reacts with 4-<br>aminoantipyrine and EMSE* to form a red purple dye. The color intensity of<br>this dye is directly proportional to the cholesterol concentration and is<br>measured photometrically. |
| | *N-ethyl-N-(3-methylphenyl)-N-succinylethylenediamine |

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Detection Limit LoB, LoD, and LoQ studies were performed based upon CLSI EP17-A2.

> LoB Protocol: One analyte free sample was tested in five-fold determinations on two analyzers over three days, for a total of N=60 determinations. Three lots of reagent were used for testing. The LoB is determined as the 95th percentile of the 60 measured values.

LoD Protocol: Five low-analyte samples were measured in singlicate on two analyzers over three days. Three lots of reagent were used for testing.

LoD was calculated as:

LoD = LoB + 1.653 x SDtot

Where:

```
SD ot = Square root [0.2 x ((SD sample 22 + (SD ample 2 + (SD sample 2 + (SD sample 2) + (SD sample 2) .
```
LoQ Protocol: A low-level sample set of five samples were tested in five replicates per sample on five days with three reagent lots, one run per day on one analyzer. The mean, the SD, and the %CV of 5 days were calculated for each sample. The mean concentration was plotted versus the %CV and LoQ is determined based on the precision at 10% CV.

The LoB, LoD, and LoQ claims represent the specifications for each.

| | Result (mg/dL) | Claim (mg/dL) |
|-----------------------|----------------|---------------|
| Limit of Blank | 0.406 | 3.87 |
| Limit of Detection | 0.99 | 3.87 |
| Limit of Quantitation | 2.28 | 3.87 |

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Precision was determined according to CLSI EP5-A2 with one analyzer and 3 Precision reagent lots using 5 human serum sample pools and two control samples (4 aliquots per run, 1 run per day, 21 days). The following results were obtained:

Repeatability Summary

| Specimen | Mean<br>mg/dL (mmol/L) | SD mg/dL<br>(mmol/L) | CV<br>(%) |
|------------------------|------------------------|----------------------|-----------|
| Precinorm L | 104 (2.69) | 0.8 (0.02) | 0.7 |
| Precipath<br>HDL/LDL-C | 191 (4.93) | 1.2 (0.03) | 0.7 |
| Human Serum 1 | 11.7 (0.302) | 0.2 (0.004) | 1.2 |
| Human Serum 2 | 113 (2.93) | 0.8 (0.02) | 0.7 |
| Human Serum 3 | 303 (7.83) | 2.3 (0.06) | 0.7 |
| Human Serum 4 | 142 (3.67) | 1.2 (0.03) | 0.7 |
| Human Serum 5 | 526 (13.6) | 4.3 (0.11) | 0.8 |

Intermediate Precision

| | Mean | SD mg/dL | CV |
|------------------------|----------------|-------------|-----|
| Specimen | mg/dL (mmol/L) | (mmol/L) | (%) |
| Precinorm L | 104 (2.69) | 2.3 (0.06) | 2.3 |
| Precipath<br>HDL/LDL-C | 194 (5.02) | 4.2 (0.11) | 2.1 |
| Human Serum 1 | 12.2 (0.316) | 0.3 (0.008) | 2.5 |
| Human Serum 2 | 117 (3.03) | 2.3 (0.06) | 2.1 |
| Human Serum 3 | 315 (8.14) | 6.2 (0.16) | 1.9 |
| Human Serum 4 | 143 (3.71) | 3.1 (0.08) | 2.1 |
| Human Serum 5 | 530 (13.7) | 10.8 (0.28) | 2.0 |

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| Analytical<br>Specificity -<br>interference<br>from common<br>drugs.<br>Simvastatin,<br>Bezafibrate,<br>and Nicotinic<br>Acid | Sixteen commonly used drugs were examined for potential interference on<br>measurement with LDL-Cholesterol Gen.<br>Two sample pools, containing a low (approximately 100 mg/dL) and high<br>(approximately 400 mg/dL) concentration of LDL are used. These sample<br>pools are divided into an appropriate number of aliquots. One aliquot is not<br>spiked with the drugs and it is used as the reference sample for LDL<br>concentration. The LDL concentration in the sample is determined with n = 3<br>measurements on a cobas c 501 analyzer. |
|-------------------------------------------------------------------------------------------------------------------------------|------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------|
| | The other sample aliquots, with either the high or low LDL concentrations,<br>are spiked with the respective amount of drug. The LDL concentration of the<br>spiked aliquots are determined in triplicate and the mean of the triplicate<br>determinations is compared to the LDL concentration determined for the<br>reference aliquot (mean of n=3). |

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| Analytical |
|------------------|
| Specificity - |
| interference |
| from common |
| drugs, |
| Simvastatin, |
| Bezafibrate, |
| and Nicotinic |
| Acid, |
| Simvastatin, |
| Bezafibrate, |
| and Nicotinic |
| Acid (continued) |

The table below summarizes the common drug interferences data:

| Drug | Highest Concentration Shown Not to Interfere<br>with LDLC3 (drug concentrations in mg/L) |
|----------------------|------------------------------------------------------------------------------------------|
| Acetylcysteine | 553 |
| Ampicillin-Na | 1000 |
| Ascorbic acid | 5000 |
| Cyclosporine | 5 |
| Cefoxitin | 2500 |
| Heparin | 5000 U |
| Levodopa | 20 |
| Methyldopa +1.5 | 20 |
| Metronidazole | 200 |
| Phenylbutazone | 400 |
| Doxycyclin | 50 |
| Acetylsalicylic Acid | 1000 |
| Rifampicin | 60 |
| Acetaminophen | 200 |
| Ibuprofen | 500 |
| Theophylline | 100 |

Additional testing was done on Simvastatin, Bezafibrate, and Nicotinic Acid. The table below summarizes the interference data:

| Drug | Highest Concentration Shown Not to Interfere<br>with LDLC3 (drug concentrations in mg/L) |
|----------------|------------------------------------------------------------------------------------------|
| Simvastatin | 16 |
| Bezafibrate | 120 |
| Nicotinic Acid | 400 |

All data passed the following acceptance criteria: Difference in recovery to the reference sample: ≤± 10%

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| Analytical<br>Specificity –<br>interference<br>from VLDL,<br>HDL,<br>Chylomicrons | The effects of interference by VLDL-cholesterol, HDL-cholesterol,<br>Chylomicrons on the LDLC3 test system was tested. |
|-----------------------------------------------------------------------------------|----------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------|
| | HDL-cholesterol: Two sample pools, containing a low and high<br>concentration of LDL were used. These sample pools were divided into two<br>aliquots. One aliquot was not spiked with HDL and it was used as the<br>reference sample for LDL concentration. The LDL concentration in the<br>sample was determined with n = 3 measurements on a cobas c 501 analyzer. |
| | The other sample aliquot, with either the high or low LDL concentrations,<br>was spiked with the respective amount of HDL. The LDL concentration of<br>the spiked aliquots were determined in triplicate and the mean of the triplicate<br>determinations was compared to the LDL concentration determined for the<br>reference aliquot (mean of n=3). |
| | The mean of the triplicate determinations was compared to the LDL<br>concentration determined for the reference aliquot (mean of n=3). |
| | VLDL-cholesterol: VLDL were isolated from fresh human serum by<br>ultracentrifugation method. Two sample pools, containing a low and high<br>concentration of LDL were used. The samples were spiked with increasing<br>amounts of VLDL fraction with increasing amounts of VLDL concentrations. |
| | The sample pools were split into two aliquots. One aliquot was spiked with<br>VLDL-fraction the second was diluted with 0.9% NaCl as the reference. |
| | The mean of the duplicate determinations is compared to the LDL<br>concentration determined for the reference aliquot (mean of n=2). |

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| Analytical<br>Specificity –<br>interference<br>from VLDL,<br>HDL,<br>Chylomicrons<br>(continued) | Chylomicrons (Triglycerides): Chylomicrons were separated from fresh<br>non-fasting human samples by centrifugation. Four sample pools**,<br>containing a low and high concentration of LDL were used. The samples<br>were spiked with increasing amounts of chylomicrons. Triglycerides<br>concentrations were measured in all samples. The sample pools were split<br>into two aliquots. One aliquot was spiked with chylomicrons the second was<br>diluted with 0.9% NaCl. The mean of the duplicate determinations was<br>compared to the LDL concentration determined for the reference aliquot<br>(mean of n=2). ** The four samples contain chylomicron concentrations ≥<br>2000 mg/dL Triglycerides. Additional samples with lower concentrations of<br>Chylomicron Triglycerides < 2000 Triglycerides were tested. |
|--------------------------------------------------------------------------------------------------|-----------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------|
| | All data passed the following acceptance criteria:<br>≤± 10% in recovery<br>VLDL-Cholesterol: ≤ 140 mg/dL<br>HDL- Cholesterol: ≤ 75 mg/dL<br>Chylomicrons: ≤ 2000 mg/dL triglycerides |
| Analytical<br>Specificity –<br>interference<br>from<br>endogenous<br>substances | The reagent was evaluated with three endogenous substances, hemoglobin,<br>Lipemia (Intralipid), and Bilirubin for potential interference with the<br>measurement of LDLC3.<br><br>One pool of human serum was spiked with the interferent. A second pool of<br>human serum contained none. The two pools were mixed in different ratios to<br>yield a dilution series with varying concentrations of the interferent.<br><br>The resulting sample series (10 dilution steps per sample) were tested in<br>triplicate and the mean values used to calculate % recovery, by comparing the<br>measured concentration to the expected concentration (which is the LDL<br>concentration when no interferent was added). |

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| Analytical<br>Specificity –<br>interference<br>from<br>endogenous<br>substances<br>(continued) | The endogenous interference data are summarized in the table. Interference was tested at two levels of reagent. | |
|------------------------------------------------------------------------------------------------|-----------------------------------------------------------------------------------------------------------------|---------------------------------------------------------------------------------------------------------------------------------|
| | no interference<br>up to | Claim as it appears in the<br>labeling. |
| Lipemia Level 1 | 1385 L index | No significant interference up to an L index of 1000 (approximate Intralipid concentration: 1000 mg/dL). |
| Lipemia Level 2 | 1967 L index | No significant interference up to an H index of 1000 (approximate hemoglobin concentration: 1000 mg/dL). |
| Hemolysis Level 1 | 1392 H index | |
| Hemolysis Level 2 | 1463 H index | No significant interference up to an I index of 60 (approximate conjugated and unconjugated bilirubin concentration: 60 mg/dL). |
| Unconjugated Bilirubin Level 1 | 77 I index | |
| Unconjugated Bilirubin Level 2 | 65 I index | |
| Conjugated Bilirubin Level 1 | 71 I index | |
| Conjugated Bilirubin Level 2 | 68 I index | |

All data passed the following acceptance criteria:

< = 10%

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Matrix Lithium-heparin, K2-EDTA and K3-EDTA are permissible anticoagulants for use Comparison with this reagent because they do not interfere with recovery of LDL-Cholesterol Gen. 3. The effect of the presence of anticoagulants on analyte recovery was determined by method comparison, obtained from samples drawn into serum and different types of plasma collection tubes (K2 EDTA, K3 EDTA, Li Heparin, and Gel Separation). One reagent lot was tested with two runs, one replicate per sample with full tubes as follows:

| | Range<br>(mg/dL) | # Samples |
|----------------|------------------|-----------|
| Gel Separation | 12.3 - 495 | 59 |
| Li-heparin | 12.3 - 495 | 59 |
| K2-EDTA | 12.3 - 495 | 57 |
| K3-EDTA | 12.3 - 495 | 59 |

Comparisons with plasma vs. serum were calculated with the following results for full tubes which passed specification :

Serum vs. Gel Separation P/B: y = 1.004x + 0.091, r = 1.000

Serum vs. Li-heparin P/B: y = 0.99x - 1.50, r = 0.999

Serum vs. K2-EDTA P/B: v = 0.98x - 0.248. r = 1.000

Serum vs. K3-EDTA P/B: y = 0.95x - 0.246, r = 0.999

{18}------------------------------------------------

Linearity Linearity was assessed according to CLSI EP6-A with one batch of reagent, in one run, and with samples measured in triplicate. Two separate dilution series differing by sample type (serum and plasma) were prepared with 14 concentrations. Dilutions were made using 0.9% NaCl. Evaluation is performed using a validated software tool provided by Roche Diagnostics Penzberg Biometry department. All the measured data of a dilution series are evaluated together in one regression analysis. The software tool plots the measured values on the y-axis against the expected values on the x-axis and calculates regressions with first-order (y=a+bx, linear model), second-order (y=a+bx+cx2; quadratic model) and third-order polynomials (y=a+bx+cx2+dx3, cubic model). In the next step it examines which of the three polynomials best describes the course of the measured data. If the first-order polynomial gives the best fit, the tested measuring range is linear. If a better fit is obtained with a second- or thirdorder polynomial, the difference between this polynomial and the first-order polynomial is calculated. In this case a third order polynomial is used. The linearity evaluation is not forced through the origin. Weighting is used: 1/conc quadratic.

> Data passed the following acceptance criteria: 3.87 mg/dL-549 mg/dL: ≤ ± 10%

Measuring range supported by Linearity Data (mg/dL)

| | Plasma | Serum |
|-----------------------------|------------|------------|
| Range tested | 3.66 - 584 | 3.53 - 565 |
| Range found | 3.66 - 584 | 3.53 - 565 |
| Recommended measuring range | 3.87 - 549 | 3.87 - 549 |

Linear Regression Equation for Serum: y = 1.0171x - 0.3682

Linear Regression Equation for Plasma: y = 1x + 0 r2 = 0.9995

{19}------------------------------------------------

| Method<br>Comparison to<br>Predicate | A total of 100 human serum samples (including 5 spiked with human LDL<br>rich serum and 2 diluted with 0.9% NaCl, range of 4.99 - 534 mg/dL) were<br>tested in singlicate with the LDLC Gen2 assay and the LDLC3 reagent on<br>cobas c 501. |
|--------------------------------------|---------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------|
| | Sample size (n) = 100 |
| | Passing/Bablok<br>y = 0.984x = 0.786 mg/dL<br>r = 0.999 |
| Conclusion | The submitted information in this premarket notification supports a<br>substantial equivalence decision. |