(35 days)
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.
The LDL-Cholesterol Gen. 3 assay is a homogeneous enzyme colorimetric assay which provides the quantitative measurement of LDL-cholesterol in human serum and plasma. Reagents are packaged in a cassette labeled with their instrument positioning R1 (Reagent 1) and R2 (Reagent 2).
R1 contains Bis-trisb) buffer: 20.1 mmol/L, pH 7.0; 4-aminoantipyrine:0.98 mmol/L; ascorbic oxidase (AOD, Acremonium spec.): ≥ 66.7 µkat/L; peroxidase (recombinant from Basidiomycetes): ≥ 166.7 µkat/L; BSA: 4.0 g/L; preservative R2 contains MOPSC) buffer: 20.1 mmol/L, pH 7.0; EMSE: 2.16 mmol/L, cholesterol esterase (Pseudomonas spec.): ≥ 33.3 µkat/L; cholesterol oxidase (recombinant from E.coli)): ≥ 31.7 µkat/L; peroxidase (recombinant from Basidiomycetes): ≥ 333.3 µkat/L; BSA: 4.0 g/L; detergents; preservative
Here's a breakdown of the acceptance criteria and study information for the LDLC3 LDL-Cholesterol Gen.3 device, organized as requested:
Acceptance Criteria and Device Performance Summary
| Performance Metric | Acceptance Criteria | Reported Device Performance |
|---|---|---|
| Limit of Blank (LoB) | Claim: 3.87 mg/dL | Result: 0.406 mg/dL (Meets acceptance criteria as it's below the claim) |
| Limit of Detection (LoD) | Claim: 3.87 mg/dL | Result: 0.99 mg/dL (Meets acceptance criteria as it's below the claim) |
| Limit of Quantitation (LoQ) | Claim: 3.87 mg/dL | Result: 2.28 mg/dL (Meets acceptance criteria as it's below the claim) |
| Drug Interference | Difference in recovery to the reference sample: ≤ ± 10% | All data passed the acceptance criteria for various common drugs, Simvastatin, Bezafibrate, and Nicotinic Acid. Specific highest concentrations shown not to interfere were reported for each drug (e.g., Acetylcysteine: 553 mg/L, Simvastatin: 16 mg/L). |
| Interference from VLDL, HDL, Chylomicrons | ≤ ± 10% in recovery for VLDL-Cholesterol: ≤ 140 mg/dL, HDL-Cholesterol: ≤ 75 mg/dL, Chylomicrons: ≤ 2000 mg/dL triglycerides | All data passed the acceptance criteria for VLDL, HDL, and Chylomicrons within their specified concentration limits. The testing methodology confirmed the device's ability to selectively measure LDL-cholesterol. |
| Endogenous Substances Interference | ≤ 10% | No significant interference was observed up to a Lipemia L index of 1000, Hemolysis H index of 1000, and Bilirubin I index of 60 (both conjugated and unconjugated). All data passed the ≤ 10% acceptance criteria. |
| Matrix Comparison | Comparisons with plasma vs. serum passed specification (details on specific regression equations and correlation coefficients are provided in the document). | 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: y = 0.98x - 0.248, r = 1.000; Serum vs. K3-EDTA P/B: y = 0.95x - 0.246, r = 0.999. All passed specification. |
| Linearity | 3.87 mg/dL - 549 mg/dL: ≤ ± 10% | For both plasma and serum: Range tested: Plasma 3.66 - 584 mg/dL, Serum 3.53 - 565 mg/dL. Range found: Plasma 3.66 - 584 mg/dL, Serum 3.53 - 565 mg/dL. Recommended measuring range: 3.87 - 549 mg/dL. Linear regression equations and r-squared values indicate good linearity (e.g., Plasma: y = 1x + 0, r2 = 0.9995). Data passed the ≤ ± 10% acceptance criteria within the recommended range. |
| Precision | Not explicitly stated as a single acceptance criterion value in the provided text, but the data indicates typical precision study results, which are generally evaluated based on CV% limits for various concentrations. The reported CVs for both repeatability and intermediate precision are low (mostly <3%), indicating good precision. | Precinorm L: Repeatability CV 0.7%, Intermediate Precision CV 2.3%; Precipath HDL/LDL-C: Repeatability CV 0.7%, Intermediate Precision CV 2.1%; Human Serum pools (various concentrations): Repeatability CVs 0.7% - 1.2%, Intermediate Precision CVs 1.9% - 2.5%. |
| Method Comparison | Not explicitly stated as a single acceptance criterion in the provided text, but determined by the correlation and agreement with the predicate device. | Passing/Bablok regression: y = 0.984x = 0.786 mg/dL, r = 0.999. This indicates very strong correlation and close agreement with the predicate device. |
Study Details
This submission describes the performance evaluation of the LDLC3 LDL-Cholesterol Gen.3 assay.
-
Sample sizes used for the test set and the data provenance:
- LoB Protocol: N=60 determinations (analyte-free sample, tested in five-fold, on two analyzers, over three days).
- LoD Protocol: Five low-analyte samples, measured in singlicate on two analyzers over three days.
- LoQ Protocol: Five low-level samples, tested in five replicates per sample on five days with three reagent lots, one run per day on one analyzer.
- Precision: 5 human serum sample pools and two control samples (4 aliquots per run, 1 run per day, 21 days).
- Drug Interferences: Two sample pools (low and high LDL concentration) for each drug, tested in triplicate. Total number of samples not explicitly stated for all drugs combined, but involves at least 2 pools x 3 replicates x 19 drugs = 114 measurements.
- Interference from VLDL, HDL, Chylomicrons: HDL: Two sample pools (low and high LDL), each divided into two aliquots, tested in triplicate. VLDL: Two sample pools (low and high LDL), spiked with increasing VLDL, measured in duplicate. Chylomicrons: Four sample pools (various chylomicron concentrations, including some ≥ 2000 mg/dL triglycerides), spiked with chylomicrons, measured in duplicate.
- Endogenous Interferences: Two human serum pools (one spiked, one unspiked) mixed in 10 dilution steps; samples tested in triplicate.
- Matrix Comparison: 59 samples for Gel Separation, 59 for Li-heparin, 57 for K2-EDTA, 59 for K3-EDTA. Samples ranged from 12.3 to 495 mg/dL. (Retrospective/Prospective not explicitly stated, but typically clinical lab studies like these are prospective collections or de-identified banked samples).
- Linearity: Two dilution series (serum and plasma), each with 14 concentrations, measured in triplicate.
- Method Comparison: 100 human serum samples (including 5 spiked and 2 diluted).
- Data Provenance: The document does not explicitly state the country of origin for the clinical samples. It refers to "human serum samples" and "human serum and plasma." This type of in-vitro diagnostic device testing would typically use samples collected under IRB-approved protocols, but the specific origins are not detailed. The studies are described in a manner consistent with prospective analytical validation.
-
Number of experts used to establish the ground truth for the test set and the qualifications of those experts:
- This is an in-vitro diagnostic device (IVD) for quantitative determination of a biomarker (LDL-cholesterol). The "ground truth" for calibrating and evaluating such devices is typically established through reference methods and certified reference materials, not through expert human interpretation of images or clinical cases. The device is being compared against a predicate device (LDL-Cholesterol plus 2nd generation).
- The document states: "This method has been standardized against the beta quantification method as defined in the recommendations in the LDL Cholesterol Method Certification Protocol for Manufacturers." This beta quantification method serves as the analytical ground truth/reference method for LDL-C measurement.
-
Adjudication method (e.g., 2+1, 3+1, none) for the test set:
- This concept of "adjudication" is not applicable to quantitative IVD studies. Adjudication methods (like 2+1, 3+1) are used in diagnostic imaging studies where multiple readers interpret cases, and discrepancies are resolved by a super-reader or consensus. For this device, direct quantitative measurements are being performed and compared to either a reference method or a predicate device.
-
If a multi-reader multi-case (MRMC) comparative effectiveness study was done, If so, what was the effect size of how much human readers improve with AI vs without AI assistance:
- No, an MRMC comparative effectiveness study was not done. This device is a standalone quantitative assay for measuring LDL-cholesterol, not an AI-powered diagnostic imaging tool that assists human readers. Therefore, the concept of human reader improvement with/without AI assistance is not relevant here.
-
If a standalone (i.e., algorithm only without human-in-the-loop performance) was done:
- Yes, the performance studies described are for the device (assay on the cobas c system) operating in a standalone manner. This is a fully automated quantitative measurement system, where the result is generated directly by the instrument, without human-in-the-loop interpretative steps that would influence the quantitative reading.
-
The type of ground truth used (expert consensus, pathology, outcomes data, etc.):
- The primary ground truth for the analytical validation is the beta quantification method as defined in the LDL Cholesterol Method Certification Protocol for Manufacturers. This is an accepted standardized reference method for truly measuring LDL-cholesterol.
- For the method comparison study, the predicate device (LDL-Cholesterol plus 2nd generation reagent) served as the comparative reference.
-
The sample size for the training set:
- This document describes an analytical validation for a quantitative in-vitro diagnostic reagent. There is no "training set" in the context of machine learning for this type of device. The assay relies on established chemical and enzymatic reactions, not machine learning algorithms trained on large datasets. The formulation of the reagent itself is based on chemical and biological principles.
-
How the ground truth for the training set was established:
- As explained above, there is no "training set" for this type of device. Therefore, the concept of establishing ground truth for a training set is not applicable. The assay's "truth" is rooted in its chemical-enzymatic mechanism and its standardization against a reference method (beta quantification).
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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 (800) 638 2041 or (301) 796-7100 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 (800) 638-2041 or (301) 796-7100 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: (317) 521-3172 Fax: (317) 521-2324 Email: noel.mencias@roche.com |
| 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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| Regulatoryinformation | Product Code | Classification | Regulation | Panel |
|---|---|---|---|---|
| LBR | Class I | 21 CFR 862.1475(Lipoprotein test system) | ClinicalChemistry75 | |
| LDLC3 LDL-Cholesterol Gen. 3 meets the limitation of exemption per 21CFR 862.9 (c)(4) - for cardiovascular risk. | ||||
| Intended use | The LDL-Cholesterol Gen. 3 assay is intended for use as an in vitro test forthe quantitative determination of LDL-Cholesterol in human serum andplasma on Roche/Hitachi cobas c systems. | |||
| Indications foruse | The LDL-Cholesterol Gen. 3 assay is an in-vitro test for the quantitativedetermination of LDL-cholesterol in human serum and plasma onRoche/Hitachi cobas c systems. Lipoprotein measurements are used in thediagnosis and treatment of lipid disorders (such as diabetes mellitus),atherosclerosis, and various liver and renal diseases. | |||
| Specialconditions foruse | For prescription use only. | |||
| Specialinstrumentrequirements | For use on the Roche/Hitachi cobas c clinical chemistry analyzer. | |||
| Continued on next page |
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| Candidatedevicedescription | The LDL-Cholesterol Gen. 3 assay is a homogeneous enzyme colorimetricassay which provides the quantitative measurement of LDL-cholesterol inhuman serum and plasma.Reagents are packaged in a cassette labeled with their instrument positioningR1 (Reagent 1) and R2 (Reagent 2). |
|---|---|
| R1 contains Bis-trisb) buffer: 20.1 mmol/L, pH 7.0; 4-aminoantipyrine:0.98 mmol/L; ascorbic oxidase (AOD, Acremoniumspec.): ≥ 66.7 µkat/L; peroxidase (recombinant from Basidiomycetes):≥ 166.7 µkat/L; BSA: 4.0 g/L; preservative R2 contains MOPSC) buffer: 20.1 mmol/L, pH 7.0; EMSE: 2.16mmol/L, cholesterol esterase (Pseudomonas spec.): ≥ 33.3 µkat/L;cholesterol oxidase (recombinant from E.coli)): ≥ 31.7 µkat/L;peroxidase (recombinant from Basidiomycetes): ≥ 333.3 µkat/L;BSA: 4.0 g/L; detergents; preservative | |
| Predicatedevice | Roche Diagnostics claims substantial equivalence to LDL-Cholesterol plus2nd generation reagent on the cobas c 501. The reagent was originally clearedin K974733 on the Boehringer Mannheim/Hitachi clinical chemistryanalyzers, and later cleared in a Special 510(k) K012287 on COBASINTEGRA. The application to the cobas c 501 analyzer was cleared onOctober 3, 2006 in K060373/A001 following the FDA Policy Document"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:LDL_C Cholesterol Plus 2ndgeneration | Candidate Device:LDLC3 LDL-Cholesterol Gen. 3 | |
| Intended Use | In vitro test for the quantitativedetermination of LDL-Cholesterol inhuman serum and plasma onRoche/Hitachi cobas c systems. | Same | |
| Sample Types | Human Serum and Plasma | Same | |
| Test Principle | Homogenous enzymatic colorimetricassav | Same | |
| Reagent Shelf LifeStability | 2-8 ℃ until expiration date | Same | |
| Reagent On-BoardStability | 12 weeks | Same | |
| Measuring Range | 0.10 - 14.2 mmol/L (3.86 - 548 mg/dL) | 3.87 -549 mg/dL | |
| Lower Limit ofMeasurement | LDL(lower detection limit) = 0.10mmol/L (3.866 mg/dL) | LoB = 0.406 mg/dLLoD = 0.99 mg/dLLoQ = 2.28 mg/dL | |
| Expected Values | Adult levels:Optimal: < 2.59 mmol/L (< 100mg/dL)Near optimal/above optimal: 2.59-3.34mmol/L (100-129 mg/dL)Borderline high: 3.37-4.12 mmol/L(130-159 mg/dL)High 4.14-4.89 mmol/L (160-189mg/dL)Very high: ≥ 4.92 mmol/L (≥ 190mg/dL) | Same | |
| Traceability | This method has been standardizedagainst the beta quantification methodas defined in the recommendations inthe LDL Cholesterol MethodCertification Protocol for Manufacturers | Same |
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Substantial Equivalence – Assay Similarities (continued)
| Assay Comparison Similarities | |||
|---|---|---|---|
| Calibrator | Calibrator for automated systems(C.f.a.s) Lipids and deionized water asthe zero calibrator. | Same | |
| (C.f.a.s. cleared for use with LDL-Cholesterol in K011658 | |||
| Calibrationfrequency | Recalibrate after reagent lot change andas required following quality controlprocedures | Same | |
| Controls | Precinorm LPrecipath HDL/LDL-CPreciControl ClinChem Multi 1PreciControl ClinChem Multi 2PreciControl ClinChem Multi 1 andPreciControl ClinChem Multi 2 werecleared for use with LDL C in 510(k)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:LDL_C Cholesterol Plus 2ndgeneration | Candidate Device:LDLC3 LDL-Cholesterol Gen. 3 |
| Test Principle | In the presence of peroxidase, thehydrogen peroxide generatedreacts with 4-aminoantipyrine andHSDA to form a purple-blue dye.The color intensity of this dye isdirectly proportional to thecholesterol concentration and ismeasured photometrically. | In the presence of peroxidase, thehydrogen peroxide generated reacts with4-aminoantipyrine and EMSE to form ared purple dye. The color intensity ofthis dye is directly proportional to thecholesterol concentration and ismeasured photometrically. |
| Reagent Composition | R1 MOPS (3-morpholinopropanesulfonic acid) buffer: 20.1 mmol/L,pH 6.5; HSDA: 0.96 mmol/L;ascorbate oxidase (Eupenicilliumspec., recombinant): ≥50 µkat/L;peroxidase (horseradish): ≥167µkat/L; preservative | R1 Bis-tris buffer: 20.1 mmol/L, pH7.0; 4-aminoantipyrine:0.98 mmol/L; ascorbic oxidase (AOD,Acremonium spec.):≥ 66.7 µkat/L; peroxidase (recombinantfrom Basidiomycetes): ≥ 166.7 µkat/L;BSA: 4.0 g/L; preservative |
| R2 MOPS (3-morpholinopropanesulfonic acid) buffer: 20.1 mmol/L,pH 6.8; MgSO4·7H2O: 8.11mmol/L; 4-aminoantipyrine: 2.46mmol/L; cholesterol esterase(Pseudomonas spec.): ≥50 µkat/L;cholesterol oxidase (Brevibacteriumspec., recombinant): ≥33.3 µkat/L;peroxidase (horseradish): ≥334µkat/L; detergent; preservative | R2 MOPS buffer: 20.1 mmol/L, pH 7.0;EMSE: 2.16 mmol/L,cholesterol esterase (Pseudomonasspec.): ≥ 33.3 µkat/L; cholesteroloxidase (recombinant from E.coli)): ≥31.7 µkat/L; peroxidase (recombinantfrom Basidiomycetes): ≥ 333.3 µkat/L;BSA: 4.0 g/L; detergents; preservative |
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| Summary oftests | The following performance data were provided in support of the substantialequivalence determination:Limit of Blank according to CLSI EP17-A2Limit of Detection according to CLSI EP17-A2Limit of Quantitation according to CLSI EP17-A2Precision according to CLSI EP5-A2Linearity according to CLSI EP6-ARerun Function Check (Post Dilution Factor)Recovery in ControlsMethod ComparisonVerification of plasma as sample materialDrug InterferencesEndogenous Interferences in serum/plasmaStability & Calibration frequencyAll performance specifications were met. |
|---|---|
| Test Principle | Cholesterol esters and free cholesterol in LDL are measured on the basis ofa cholesterol enzymatic method using cholesterol esterase and cholesteroloxidase in the presence of surfactants which selectively solubilizes onlyLDL. The enzyme reactions to the lipoproteins other than LDL are inhibitedby surfactants and a sugar compound. Cholesterol in HDL, VLDL andchylomicron is not determined. |
| Cholesterol esters are broken down quantitatively into free cholesterol andfatty acids by cholesterol esterase. | |
| In the presence of oxygen, cholesterol is oxidized by cholesterol oxidase toΔ4-cholestenone and hydrogen peroxide. | |
| In the presence of peroxidase, the hydrogen peroxide generated reacts with 4-aminoantipyrine and EMSE* to form a red purple dye. The color intensity ofthis dye is directly proportional to the cholesterol concentration and ismeasured 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 | Meanmg/dL (mmol/L) | SD mg/dL(mmol/L) | CV(%) |
|---|---|---|---|
| Precinorm L | 104 (2.69) | 0.8 (0.02) | 0.7 |
| PrecipathHDL/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 |
| PrecipathHDL/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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| AnalyticalSpecificity -interferencefrom commondrugs.Simvastatin,Bezafibrate,and NicotinicAcid | Sixteen commonly used drugs were examined for potential interference onmeasurement with LDL-Cholesterol Gen.Two sample pools, containing a low (approximately 100 mg/dL) and high(approximately 400 mg/dL) concentration of LDL are used. These samplepools are divided into an appropriate number of aliquots. One aliquot is notspiked with the drugs and it is used as the reference sample for LDLconcentration. The LDL concentration in the sample is determined with n = 3measurements on a cobas c 501 analyzer. |
|---|---|
| The other sample aliquots, with either the high or low LDL concentrations,are spiked with the respective amount of drug. The LDL concentration of thespiked aliquots are determined in triplicate and the mean of the triplicatedeterminations is compared to the LDL concentration determined for thereference 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 Interferewith 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 Interferewith 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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| AnalyticalSpecificity –interferencefrom VLDL,HDL,Chylomicrons | The effects of interference by VLDL-cholesterol, HDL-cholesterol,Chylomicrons on the LDLC3 test system was tested. |
|---|---|
| HDL-cholesterol: Two sample pools, containing a low and highconcentration of LDL were used. These sample pools were divided into twoaliquots. One aliquot was not spiked with HDL and it was used as thereference sample for LDL concentration. The LDL concentration in thesample was determined with n = 3 measurements on a cobas c 501 analyzer. | |
| The other sample aliquot, with either the high or low LDL concentrations,was spiked with the respective amount of HDL. The LDL concentration ofthe spiked aliquots were determined in triplicate and the mean of the triplicatedeterminations was compared to the LDL concentration determined for thereference aliquot (mean of n=3). | |
| The mean of the triplicate determinations was compared to the LDLconcentration determined for the reference aliquot (mean of n=3). | |
| VLDL-cholesterol: VLDL were isolated from fresh human serum byultracentrifugation method. Two sample pools, containing a low and highconcentration of LDL were used. The samples were spiked with increasingamounts of VLDL fraction with increasing amounts of VLDL concentrations. | |
| The sample pools were split into two aliquots. One aliquot was spiked withVLDL-fraction the second was diluted with 0.9% NaCl as the reference. | |
| The mean of the duplicate determinations is compared to the LDLconcentration determined for the reference aliquot (mean of n=2). |
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| AnalyticalSpecificity –interferencefrom VLDL,HDL,Chylomicrons(continued) | Chylomicrons (Triglycerides): Chylomicrons were separated from freshnon-fasting human samples by centrifugation. Four sample pools**,containing a low and high concentration of LDL were used. The sampleswere spiked with increasing amounts of chylomicrons. Triglyceridesconcentrations were measured in all samples. The sample pools were splitinto two aliquots. One aliquot was spiked with chylomicrons the second wasdiluted with 0.9% NaCl. The mean of the duplicate determinations wascompared to the LDL concentration determined for the reference aliquot(mean of n=2). ** The four samples contain chylomicron concentrations ≥2000 mg/dL Triglycerides. Additional samples with lower concentrations ofChylomicron Triglycerides < 2000 Triglycerides were tested. |
|---|---|
| All data passed the following acceptance criteria:≤± 10% in recoveryVLDL-Cholesterol: ≤ 140 mg/dLHDL- Cholesterol: ≤ 75 mg/dLChylomicrons: ≤ 2000 mg/dL triglycerides | |
| AnalyticalSpecificity –interferencefromendogenoussubstances | The reagent was evaluated with three endogenous substances, hemoglobin,Lipemia (Intralipid), and Bilirubin for potential interference with themeasurement of LDLC3.One pool of human serum was spiked with the interferent. A second pool ofhuman serum contained none. The two pools were mixed in different ratios toyield a dilution series with varying concentrations of the interferent.The resulting sample series (10 dilution steps per sample) were tested intriplicate and the mean values used to calculate % recovery, by comparing themeasured concentration to the expected concentration (which is the LDLconcentration when no interferent was added). |
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| AnalyticalSpecificity –interferencefromendogenoussubstances(continued) | The endogenous interference data are summarized in the table. Interference was tested at two levels of reagent. | |
|---|---|---|
| no interferenceup to | Claim as it appears in thelabeling. | |
| 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(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
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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
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| MethodComparison toPredicate | A total of 100 human serum samples (including 5 spiked with human LDLrich serum and 2 diluted with 0.9% NaCl, range of 4.99 - 534 mg/dL) weretested in singlicate with the LDLC Gen2 assay and the LDLC3 reagent oncobas c 501. |
|---|---|
| Sample size (n) = 100 | |
| Passing/Babloky = 0.984x = 0.786 mg/dLr = 0.999 | |
| Conclusion | The submitted information in this premarket notification supports asubstantial equivalence decision. |
§ 862.1475 Lipoprotein test system.
(a)
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.(b)
Classification. Class I (general controls). The device is exempt from the premarket notification procedures in subpart E of part 807 of this chapter subject to § 862.9.