NMR LIPOPROFILE (R) TEST AND NMR PROFILER

{0} 1 # 510(k) SUBSTANTIAL EQUIVALENCE DETERMINATION DECISION SUMMARY ASSAY ONLY TEMPLATE A. 510(k) Number: k111516 B. Purpose for Submission: Modification of a previously cleared device to change software architecture for NMR LipoProfile Test (LDL-P, HDL-C and triglycerides) and NMR curve fit algorithm for LDL-P C. Measurand: LDL-P (low density lipoprotein particle number), HDL cholesterol, triglycerides D. Type of Test: Nuclear magnetic resonance (NMR) E. Applicant: LipoScience Inc. F. Proprietary and Established Names: NMR LipoProfile Test G. Regulatory Information: | Product Code | Classification | Regulation Section | Panel | | --- | --- | --- | --- | | MRR | I, meets limitations per 21 CFR 862.9 (c)(4) | 21 CFR §862.1475 – Lipoprotein test system | Chemistry (75) | | LBS | I, meets limitations per 21 CFR 862.9 (c)(4) | 21 CFR §862.1175 – Cholesterol test system | Chemistry (75) | | CDT | I, meets limitations per 21 CFR 862.9 (c)(4) | 21 CFR §862.1705 – Triglyceride test system | Chemistry (75) | H. Intended Use: 1. Intended use(s): See Indications for use below. {1} 2. Indication(s) for use: The NMR LipoProfile® test, when used with the NMR Profiler, an automated NMR spectrometer, measures lipoprotein particles to quantify LDL particle number (LDL-P), HDL cholesterol (HDL-C), and triglycerides in human serum and plasma using nuclear magnetic resonance (NMR) spectroscopy. LDL-P and these NMR-derived concentrations of HDL-C and triglycerides are used in conjunction with other lipid measurements and clinical evaluation to aid in the management of lipoprotein disorders associated with cardiovascular disease. This test is performed and provided as a service by LipoScience Laboratory. 3. Special conditions for use statement(s): For prescription use only. 4. Special instrument requirements: This device is cleared as a service (in-house). Samples are sent to Liposcience Laboratories to be run on the NMR Profiler, using software version Gen2.1. I. Device Description: The test system includes a 400 MHz proton NMR spectrometer interfaced with a commercial sample handler, an analysis server containing the software to analyze digitized spectral data, and the following reagents: - NMR Diluent - aqueous solution containing Na₂EDTA (5.0 mM), CaCl₂ (1.0 mM), KCL (120 mM), Na₂HPO₄ (50 mM), NaN₃ (0.02%), pH 7.4 - NMR Wash - Triton X-100-0.1% v/v, Liqui Nox 0.1% v/v in deionized water, pH 10.0 - NMR Calibrator - aqueous solution of Trimethyl Acetate (TMA) disodium salt (15.0 mM) containing Na₂EDTA (5.0 mM), CaCl₂ (3.0 mM), KCl (120 nM), D₂O 10% v/v - NMR LipoProfile Quality Control material - two levels of pooled human serum-based control material, (Control A and Control B), with pre-determined target ranges. The control materials contain human source material. Each donor unit is tested by FDA – approved methods and found non-reactive for hepatitis B surface antigen (HBsAg), antibody to hepatitis C, and antibody to HIV-1/2, all products using human source material should be handled as potentially infectious, because no test method can offer complete assurance that infectious agents are absent. J. Substantial Equivalence Information: 1. Predicate device name(s): NMR Profiler and NMR Lipoprofile Assay 2. Predicate K number(s): k063841 {2} 3. Comparison with predicate: | Similarities | | | | --- | --- | --- | | Item | Predicate device (k063841) | Proposed device | | Indications for use | Measures lipoprotein particles to quantify LDL particle number (LDL-P), HDL cholesterol (HDL-C), and triglycerides in human serum and plasma using nuclear magnetic resonance (NMR) spectroscopy. LDL-P and these NMR-derived concentrations of HDL-C and triglycerides are used in conjunction with other lipid measurements and clinical evaluation to aid in the management of lipoprotein disorders associated with cardiovascular disease. | Same | | Specimen type | Serum and plasma | Same | | Reference range study | 5^{th} through 95^{th} percentile values for LDL-P obtained for samples from the Multi-Ethnic Study of Athlerosclerosis | Same (Ranges from predicate transferred to new device via CLSI Guideline C28-A2; see section M5 below) | {3} | Differences | | | | --- | --- | --- | | Item | Predicate device (k063841) | Proposed device | | Software version that includes algorithm used to curve fit to the NMR peak and calculate the LDL-P results and software architecture | LP2 | Gen2.1 | | Software architecture | Calculation of all three analytes (LDL-P, HDL concentration and triglycerides) performed by one algorithm in one software module. Changes to the algorithm result in changes in calculations for all analytes. | Modular (separate software modules for each analyte that allows for modules to change and new modules to be added with no impact to the signal generation or to other unmodified analytes) | K. Standard/Guidance Document Referenced (if applicable): - CLSI Guideline EP5-A2: Evaluation of Precision Performance of Quantitative Measurement Methods - CLSI Guideline EP 17-A: Protocols for Determination of Limits of Detection and Limits of Quantitation - CLSI Guideline EP6-A: Evaluation of the Linearity of Quantitative Measurement Procedures: A Statistical Approach - CLSI Guideline EP9-A2: Method Comparison and Bias Estimation Using Patient Samples - CLSI Protocol EP7-A2: Interference Testing in Clinical Chemistry - CLSI Guideline C28-A2: How to Define and Determine Reference Intervals in the Clinical Laboratory L. Test Principle: The NMR LipoProfile® test and NMR Profiler involves measurement of the 400 MHz proton NMR spectrum of a plasma/serum sample, deconvolution of the composite signal at approximately 0.8 ppm to produce signal amplitudes of the lipoprotein subclasses that contribute to the composite plasma/serum signal, and conversion of these subclass signal amplitudes to lipoprotein subclass concentrations. The ~0.8 ppm plasma NMR signal arises from the methyl group protons of the lipids {4} carried in the LDL, HDL and VLDL subclasses of varying diameters. The NMR signals from the various lipoprotein subclasses have unique and distinctive frequencies and line shapes, each of which is accounted for in the deconvolution analysis model. Each subclass signal amplitude is proportional to the number of subclass particles emitting the signal, which enables subclass particle concentrations to be calculated from the subclass signal amplitudes derived from the spectral deconvolution analysis. LDL subclass particle concentrations, in units of nanomoles of particles per liter (nmol/L), are summed to give the reported total LDL particle concentration (LDL-P). By employing conversion factors assuming that the various lipoprotein subclass particles have cholesterol and triglyceride contents characteristic of normolipidemic individuals, HDL cholesterol and triglyceride concentrations are also derived. ## M. Performance Characteristics (if/when applicable): ### 1. Analytical performance: #### a. Precision/Reproducibility: Within-run and within-laboratory precision were evaluated in accordance with the methods defined in the CLSI Guideline EP5-A, “Evaluation of Precision Performance of Clinical Chemistry Devices.” Three serum pools with different analyte concentrations were prepared and tested in multiple runs over multiple days. For the Within-Lab, multiple instruments, operators and reagent lots were incorporated into the testing and a variance component analysis was conducted to estimate the individual sources of the total system variability. **Within-run precision** - A single run of 20 replicates for the low and the medium pool was conducted on one instrument and a single run of 20 replicates for the high pool was conducted on a second NMR instrument. A single operator conducted all three runs. LDL-P (nmol/L) | Pool | N | Mean | SD | SD 95% CI | CV | | --- | --- | --- | --- | --- | --- | | Low | 20 | 908 | 45.4 | 34.5 – 66.3 | 5.0% | | Medium | 20 | 1493 | 64.8 | 49.3 – 94.7 | 4.3% | | High | 19* | 1967 | 72.8 | 55.0 – 107.6 | 3.7% | *One replicate of the high pool produced no data due to instrument failure (sample delivery failure) and therefore only 19 replicates were analyzed. {5} 6 HDL-C (mg/dL) | Pool | N | Mean | SD | SD 95% CI | CV | | --- | --- | --- | --- | --- | --- | | Low | 20 | 23.7 | 0.5 | 0.4 - 0.7 | 2.0% | | Medium | 19* | 54.9 | 1.0 | 0.8 - 1.5 | 1.9% | | High | 20 | 95.1 | 0.9 | 0.6 - 1.2 | 0.9% | Triglycerides (mg/dL) | Pool | N | Mean | SD | SD 95% CI | CV | | --- | --- | --- | --- | --- | --- | | Low | 20 | 81.0 | 2.1 | 1.6 - 3.1 | 2.6% | | Medium | 19* | 140.6 | 2.5 | 1.9 - 3.7 | 1.8% | | High | 20 | 649.5 | 8.7 | 6.6 - 12.7 | 1.3% | * One replicate of the medium pool produced no data due to an instrument failure (sample delivery failure) and therefore, only has 19 replicates analyzed. Within-laboratory precision – Two runs per day with two replicates per run for three pools were tested on three instruments for a total of 20 testing days. Five operators conducted the study over the 20 testing days. Additionally, six lots of NMR Diluent 1 were used over the 20 days of testing. LDL-P (nmol/L): | Pool | N | Mean | SD | SD 95% CI | CV | | --- | --- | --- | --- | --- | --- | | Low | 240 | 932.6 | 64.6 | 58.8 - 71.7 | 6.9% | | Medium | 240 | 1539.3 | 86.9 | 77.3 – 99.3 | 5.6% | | High | 240 | 2046.1 | 111.8 | 99.3 – 127.9 | 5.5% | {6} 7 HDL-C (mg/dL): | Pool | N | Mean | SD | SD 95% CI | CV | | --- | --- | --- | --- | --- | --- | | Low | 240 | 23.5 | 0.9 | 0.8 – 1.0 | 3.9% | | Medium | 240 | 56.5 | 1.3 | 1.2-1.6 | 2.4% | | High | 240 | 96.2 | 2.5 | 2.1 – 2.9 | 2.6% | Triglycerides (mg/dL): | Pool | N | Mean | SD | SD 95% CI | CV | | --- | --- | --- | --- | --- | --- | | Low | 240 | 78.0 | 2.9 | 2.6 – 3.3 | 3.7% | | Medium | 240 | 146.0 | 4.2 | 3.6 – 4.9 | 2.8% | | High | 240 | 627.5 | 20.7 | 17.7 – 24.8 | 3.3% | b. Linearity/assay reportable range: The claimed measuring ranges of this device are: LDL-P: 300 - 3500 nmol/L HDL-C: 7 - 140 mg/dL Triglycerides: 5 - 1100 mg/dL LDL-P: The dilution series consisted of eleven samples that were prepared by combining selected de-identified, residual specimens from LipoScience’s commercial laboratory. The samples covered the range from 184 nmol/L to 3868 nmol/L. All the pools were run on the same day in 4 replicates. The study was conducted in a single run on one instrument. Significance of the terms for both the linear and polynomial regression was examined. {7} | R=0.9987, R²=0.9974 | | | | | --- | --- | --- | --- | | Linear Term | Coefficient | SE | p | | Intercept | -11.9944 | 19.4903 | 0.5424 | | X | 0.9843 | 0.0086 | < 0.0001 | | R=0.9990, R²=0.9980 | | | | | Non-Linear Term | Coefficient | SE | p | | Intercept | 39.4779 | 23.4440 | 0.1016 | | X | 0.8929 | 0.0293 | < 0.0001 | | X² | 0.0000 | 0.0000 | 0.0027 | In the polynomial regression, the $\mathrm{X}^2$ term was significant (p=0.0027). Given the significance of the nonlinear term, acceptance criteria for allowable nonlinearity were evaluated. Samples 5-11 met the sponsor's pre-determined allowable nonlinearity criteria (10%). The low concentration samples 2-3, compared to the absolute nonlinearity criteria of 52.1 nmol/L, meet the sponsor's allowable nonlinearity criteria. Therefore the linearity observed for LDL-P in this study supports the claimed measuring range of 300 to 3500 nmol/L. HDL-C and triglycerides: Twelve concentration levels were prepared across the range of the assays by serial dilutions of the source pools and tested. The study was conducted in a single run on one instrument and each level was tested in replicates of four. For HDL-C, the samples covered the range of 6.3 - 205 mg/dL; for triglycerides the samples covered the range 5.4 - 1880 mg/dL. Linear regression analysis yielded the following results: HDL-C: $\mathrm{R}^2 = 0.9998$ $\mathrm{y} = 1.004\mathrm{x} - 0.5956$ Triglycerides: $\mathrm{R}^2 = 0.9998$ $\mathrm{y} = 1.006 + 2.057$ c. Traceability, Stability, Expected values (controls, calibrators, or methods): Reagents, calibrators and controls are unchanged from the previously cleared {8} device (k063841). d. Detection limit: The lower ends of the measuring ranges for LDL-P, HDL-C and triglycerides were determined by linearity, above in 2b, and unchanged from the predicate (k063841): LDL-P: 300 mmol/L HDL-C: 7 mg/dL Triglycerides: 5 mg/dL e. Analytical specificity: A new interference study was performed to evaluate whether substances could be potential interferents of the device and its modified software. In addition, two new therapeutics for the intended use population became available and were also evaluated in the new study. In order to evaluate potential interferents with the new version of the LDL-P software Gen2.1, LipoScience employed the following study design: first, identify potentially interfering substances that show resonance(s) in the vicinity of lipoprotein resonances. Each potential interferent was diluted in the appropriate solvent and analyzed on the NMR Profiler to determine if it demonstrated peak(s) in the 0.7 – 1.0 ppm region. Five endogenous substances and 22 drugs were screened in this step using concentrations in accordance to CLSI EP7-A2. Second, once a potentially interfering substance was identified, a spiking study was completed where the substance was added to sample pools containing two different levels of LDL-P, HDL-C and triglycerides for a paired difference test. 9 {9} 10 Endogenous Exogenous (OTC drugs, etc.) | Potential Interferent | Test Concentration | Potential Interferent | Test Concentration | | --- | --- | --- | --- | | Hemoglobin | 2 mg/mL | Simvastatin | 48 μg/mL | | Bilirubin, unconj. | 200 μg/mL | Fenofibrate | 45 μg/mL | | Creatinine | 50 μg/mL | Nicotinic Acid Sodium salt | 1.2 mg/mL | | Urea | 2.6 mg/mL | Acetylsalicylic acid | 652 μg/mL | | Uric acid | 235 μg/ml | Acetaminophen | 200 μg/mL | | | | Naproxen Sodium | 547 μg/mL | | | | Ibuprofen Sodium salt | 210^{1}, 70^{2} μg/mL | | | | Piroxicam | 60 μg/mL | | | | Hydrochlorothiazide | 6.0 μg/mL | | | | Triamterene | 8.9 μg/mL | | | | Pravastatin | 48 μg/mL | | | | Furosemide | 60 μg/mL | | | | Metoprolol tartrate | 6.4 μg/mL | | | | Nifedipine | 400 μg/mL | | | | Enalaprilat Dihydrate | 0.3 μg/mL | | | | Hydralazine hydrochloride | 180 μg/mL | | | | Isosorbide dinitrate (lactose mixture 2:3) | 375 μg/mL | | | | Clopidogrel hydrogensulfate | 360 μg/mL | | | | Glipizide | 2.0 μg/mL | | | | Metformin Hydrochloride | 600 μg/mL | | | | Pioglitazone hydrochloride | 27 μg/mL | | | | Atorvastatin | 48 μg/mL | 1 For LDL-P and HDL concentration 2 test results for triglycerides were ~4 to 6% lower at the 140 and 210 μg/mL concentrations of ibuprofen f. Assay cut-off: Not applicable. 2. Comparison studies: a. Method comparison with predicate device: **LDL-P**: The comparison was conducted in agreement with CLSI EP9-A2: Method Comparison and Bias Estimation Using Patient Samples. The study involved testing singlicates followed by statistical analysis of 1600 freshly-collected clinical de-identified specimens across the range of the LDL-P test. Bias was calculated for LDL-P from the linear regression line at the medical decision limits of 1000, 1300 and 1600 nmol/L as well. 1555 samples are included in the final analysis (44 samples were outside of the claimed {10} measuring range; one sample failed QC check due to an instrument shim error and could not be repeated due to insufficient sample volume). The range of the samples was 315 to 3497 nmol/L. ## Linear regression/least squares analysis: $$ r = 0.970 $$ Slope = 0.98 95% CI: 0.97 – 0.99 Intercept = 46.42 95% CI: 22.44 – 70.40 ## Deming Regression analysis: Slope = 1.01 95% CI: 1.00 - 1.03 Intercept = -7.97 95% CI: -31.45 – 15.50 In addition to conducting a method comparison for the overall data set, the data were classified into the three following segments based on LDL-P values; (1) results below the medical decision limits of 1000 nmol/L, (2) results between 1000 and 1600 nmol/L and (3) results above 1600 nmol/L. For each segment the bias was determined. The determined % Bias for each segment was less than the acceptable allowable bias (&lt; 5% for each level). Observed LDL-P Bias per linear regression at Medical Decision Limits | | Linear regression | | Deming regression | | | --- | --- | --- | --- | --- | | LDL-P (nmol/L) | Absolute Bias | % Bias | Absolute Bias | % Bias | | 1000 | 28.0 | 2.8% | 3.9 | 0.4% | | 1300 | 22.6 | 1.7% | 7.5 | 0.6% | | 1600 | 17.0 | 1.1% | 11.1 | 0.7% | ## HDL-C and triglycerides: A total of 1600 freshly-collected clinical de-identified specimens from the LipoScience commercial lab were evaluated. The performance of these samples run on the proposed device was compared to results given by the predicate (k063841). $$ \mathrm{HDL - C:} $$ $$ y = 1.00x + 0.03 $$ $$ R = 0.999 $$ $$ \text{Range} = 12.0 - 140.0 \, \mathrm{mg/dL} $$ Triglycerides: n = 1597 (two samples outside the measuring range; one sample failed as indicated above under LDL-P method comparison) $$ y = 1.00x + 1.17 $$ $$ r = 1.000 $$ $$ \text{Range} = 20 - 925 \, \mathrm{mg/dL} $$ {11} b. Matrix comparison: The various specimen tubes recommended by the manufacturer for use with this assay are serum drawn in gel barrier NMR LipoTubes (Greiner, Inc. Part #456293), or red-top plain serum collection tubes; or plasma drawn into EDTA or heparin collection tubes. A study was conducted to validate the equivalence of NMR LipoProfile results using these serum and plasma specimens. Blood was collected in the specified collection tubes and immediately tested. The study comparing samples collected in serum tubes (NMR LipoTube, reference) to plain serum (red-top), EDTA plasma (purple top) or Na Heparin plasma (green top) tubes tested individual specimens from 30 volunteers. Samples tested spanned most of the assay range. Average biases observed ranged between 2.6 to 6.1% for LDL-P. No concentration-dependent trends were observed. 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: The sponsor used procedures in CLSI Guideline C28-A2 to bridge the transference of a reference range established with the predicate method in k063841 to the proposed device. For the predicate devices, in order to determine the distribution of LDL-P levels expected in a representative sampling of the general population, plasma samples (n=5,362) were analyzed from apparently healthy men and women (mean age 61, ranging from 44 to 84 years) enrolled in the Multi-Ethnic Study of Atherosclerosis (MESA), a large epidemiologic study sponsored by the National Heart, Lung, and Blood Institute. Results were presented as percentiles. In the sponsor’s bridging study, the plasma samples from the MESA study were re-tested. Statistical analysis demonstrated that the percentiles determined for the predicate are also valid for the proposed device. {12} | | All (n=5,362) | Men (n=2,529) | Women (n=2,833) | | --- | --- | --- | --- | | Percentile | LDL-P (nmol/L) | LDL-P (nmol/L) | LDL-P (nmol/L) | | 5 | 770 | 800 | 760 | | 10 | 870 | 900 | 850 | | 20 | 1000 | 1040 | 970 | | 30 | 1100 | 1150 | 1060 | | 40 | 1190 | 1250 | 1150 | | 50 | 1280 | 1330 | 1230 | | 60 | 1380 | 1430 | 1330 | | 70 | 1480 | 1530 | 1440 | | 80 | 1610 | 1640 | 1570 | | 90 | 1790 | 1820 | 1760 | | 95 | 1980 | 1990 | 1970 | HDL concentration and triglycerides: The proposed device has similar reference values as the predicate. The reference values for patient classification have been recommended by the NCEP for HDL cholesterol and triglycerides for the assessment and management of CVD risk. Each laboratory should verify the validity of these reference values for the population it serves. | HDL Cholesterol, mg/dL Classification | | | --- | --- | | Low | High | | <40 | ≥60 | | Triglycerides, mg/dL Classification | | | | | --- | --- | --- | --- | | Normal | Borderline-high | High | Very high | | < 150 | 150-199 | 200-499 | ≥500 | # 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.