PLAC TEST REAGENT KIT,CALIBRATOR KIT, LP-PLA2 CONTROL KIT, MODEL(S) 90107,90109,90109

K072599 · Diadexus, Inc. · NOE · Dec 20, 2007 · Immunology

Device Facts

• Record ID: K072599
• Device Name: PLAC TEST REAGENT KIT,CALIBRATOR KIT, LP-PLA2 CONTROL KIT, MODEL(S) 90107,90109,90109
• Applicant: Diadexus, Inc.
• Product Code: NOE · Immunology
• Decision Date: Dec 20, 2007
• Decision: SESE
• Submission Type: Traditional
• Regulation: 21 CFR 866.5600
• Device Class: Class 2

Indications for Use

The PLAC® Test Reagent Kit is a turbidimetric immunoassay for the quantitative determination of Lp-PLA₂ (lipoprotein-associated phospholipase A₂) in human plasma or serum on automated clinical chemistry analyzers, to be used in conjunction with clinical evaluation and patient risk assessment as an aid in predicting risk for coronary heart disease, and ischemic stroke associated with atherosclerosis. The PLAC® Test Calibrator Kit is intended to establish points of reference that are used in the determination of values in the measurement of Lp-PLA₂ by the PLAC® Test Reagent Kit. The Lp-PLA₂ Control Kit is intended for use as a quality control tool to monitor the performance within the clinical range of the PLAC® Test Reagent Kit, a turbidimetric immunoassay for the quantitative determination of Lp-PLA₂.

Device Story

The PLAC® Test is a quantitative turbidimetric immunoassay for measuring Lp-PLA₂ levels in human serum or plasma. The device utilizes latex microparticles coated with mouse monoclonal antibodies (2C10 and 4B4) that bind to Lp-PLA₂ in the sample, causing agglutination. The degree of turbidity is measured by an automated clinical chemistry analyzer (validated on Hitachi® 917) at 570 nm. The concentration is interpolated from a standard curve generated by a five-level calibrator set using a spline curve fit. The test is performed in professional clinical laboratories. Results are used by clinicians alongside other clinical evaluations to assess patient risk for coronary heart disease and ischemic stroke. The device provides a quantitative value that assists in identifying patients at higher risk, potentially guiding clinical decision-making regarding cardiovascular health management.

Clinical Evidence

No new clinical data were generated. Substantial equivalence is supported by analytical performance data, including a minimum detection limit of 4.0 ng/mL, intra-assay precision (1.6-2.4% CV), total precision (1.8-3.2% CV), and linearity (96-472 ng/mL). A method comparison study between the turbidimetric immunoassay and the predicate ELISA method showed a correlation coefficient of r = 0.95 with a slope of 1.02.

Technological Characteristics

Turbidimetric immunoassay using latex microparticles coated with mouse monoclonal antibodies (2C10 and 4B4). Reagents: R1 (Tris-based buffer), R2 (coated latex beads). Detection: Automated clinical chemistry analyzer (570 nm). Calibration: 5-level recombinant Lp-PLA₂. Linearity: 7-500 ng/mL. Stability: 16 months at 2-8°C. Software: Spline curve fitting for concentration interpolation.

Indications for Use

Indicated for use in patients as an aid in predicting risk for coronary heart disease and ischemic stroke associated with atherosclerosis. Used for quantitative determination of Lp-PLA2 in human plasma or serum.

Regulatory Classification

Identification

A low-density lipoprotein immunological test system is a device that consists of the reagents used to measure by immunochemical techniques the low-density lipoprotein in serum and other body fluids. Measurement of low-density lipoprotein in serum may aid in the diagnosis of disorders of lipid (fat) metabolism and help to identify young persons at risk from cardiovascular diseases.

Predicate Devices

• diaDEXUS PLAC Test (k062234)
• diaDEXUS PLAC Test (k050523)
• diaDEXUS PLAC Test (k030477)

Submission Summary (Full Text)

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

A. 510(k) Number:
k072599

B. Purpose for Submission:
Clearance of new methodology (turbidimetric immunoassay) for previously cleared device (enzyme immunoassay)

C. Measurand:
Lipoprotein-Associated Phospholipase A₂ (Lp-PLA₂)

D. Type of Test:
Quantitative turbidimetric immunoassay

E. Applicant:
diaDexus, Inc.

F. Proprietary and Established Names:
PLAC® Test Reagent Kit
PLAC® Test Calibrator Kit
Lp-PLA₂ Control Kit

G. Regulatory Information:

1. Regulation section:

| Product Code | Classification | Regulation Section | Panel |
| --- | --- | --- | --- |
| NOE-test, system, immunoassay, lipoprotein-associated phospholipase a2 | Class II | 21 CFR 866.5600 | 82 Immunology |
| JIT- calibrator, secondary | Class II | 21 CFR 866.1150 | 75 Chemistry |
| JJX- quality control material | Class I | 21 CFR 862.1660 | 75 Chemistry |

H. Intended Use:

1. Intended use(s):

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See indications for use below.

2. Indication(s) for use:
The PLAC® Test Reagent Kit is a turbidimetric immunoassay for the quantitative determination of Lp-PLA₂ (lipoprotein-associated phospholipase A₂) in human plasma or serum on automated clinical chemistry analyzers, to be used in conjunction with clinical evaluation and patient risk assessment as an aid in predicting risk for coronary heart disease, and ischemic stroke associated with atherosclerosis.

The PLAC® Test Calibrator Kit is intended to establish points of reference that are used in the determination of values in the measurement of Lp-PLA₂ by the PLAC® Test Reagent Kit.

The Lp-PLA₂ Control Kit is intended for use as a quality control tool to monitor the performance within the clinical range of the PLAC® Test Reagent Kit, a turbidimetric immunoassay for the quantitative determination of Lp-PLA₂.

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

4. Special instrument requirements:
The PLAC Test was validated on the Roche Hitachi® 917.

I. Device Description:
- The diaDexus PLAC® Test assay consists of separately packaged reagents, calibrators and controls for the measurement of Lp-PLA₂ in serum or plasma. The PLAC® Test Reagent Kit consists of two reagents. R1 is a tris-based buffer solution and R2 is a suspension of latex microparticles coated with mouse monoclonal antibodies specific to Lp-PLA₂ (2C10 and 4B4).
- The PLAC® Test Calibrator Kit is a five level set of Lp-PLA₂ calibrators made with recombinant Lp-PLA₂ in a protein stabilizing buffer and used to calibrate the PLAC assay.
- The Lp-PLA₂ Control Kit is a two level set of Lp-PLA₂ controls made with recombinant Lp-PLA₂ in a protein stabilizing buffer.

J. Substantial Equivalence Information:

1. Predicate device name(s):
diaDEXUS PLAC Test

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2. Predicate K number(s):

k062234, k050523 and k030477

3. Comparison with predicate:

| Similarities | | |
| --- | --- | --- |
| | Predicate PLAC® Test k062234 | Current Device |
| Intended Use | REAGENT KIT The diaDexus PLAC® Test is an enzyme immunoassay for the quantitative determination of Lp-PLA2 (lipoprotein-associated phospholipase A2) in human plasma and serum, to be used in conjunction with clinical evaluation and patient risk assessment as an aid in predicting risk for coronary heart disease, and ischemic stroke associated with atherosclerosis. (Calibrators and controls were included in kit.) | REAGENT KIT The PLAC® Test Reagent Kit is a turbidimetric immunoassay for the quantitative determination of Lp-PLA2 (lipoprotein-associated phospholipase A2) in human plasma or serum on automated clinical chemistry analyzers, to be used in conjunction with clinical evaluation and patient risk assessment as an aid in predicting risk for coronary heart disease, and ischemic stroke associated with atherosclerosis. CALIBRATOR KIT The PLAC® Test Calibrator Kit is intended to establish points of reference that are used in the determination of values in the measurement of Lp-PLA2 by the PLAC® Test Reagent Kit. CONTROL KIT The Lp-PLA2 Control Kit is intended for use as a quality control tool to monitor the performance within the clinical range of the PLAC® Test Reagent Kit, a turbidimetric immunoassay for the quantitative determination of Lp-PLA2. |
| Analyte | Lp-PLA2 | Same |
| Laboratory Environment | Professional laboratory | Same |

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| Differences | | |
| --- | --- | --- |
| | Predicate PLAC® Test k062234 | Current Device |
| Sample | Serum | Serum, EDTA-plasma, heparin-plasma |
| Reagent Components | Dual monoclonal antibody sandwich ELISA: · anti-Lp-PLA2 mAb (2C10) coated strip wells · Wash Buffer · Enzyme Conjugate : anti-Lp-PLA2 mAb (4B4)-HRP · TMB Substrate Solution · Stop Solution | Two-reagent system: · R1: Tris-based buffer solution · R2: Suspension of anti-Lp-PLA2 (mAbs 2C10 and 4B4) coated latex beads |
| Calibration | Six calibrators made with recombinant Lp-PLA2 in a buffered protein matrix (included in ELISA kit) | Five calibrators made with recombinant Lp-PLA2 in a buffered protein matrix (sold separately) |
| Calibration Conc. | 0, 50, 100, 250, 500, 1000 ng/mL | 0, 50, 100, 250, 500 ng/mL |
| Methodology | Microplate Enzyme immunoassay (ELISA) | Latex particle-enhanced turbidimetric immunoassay (particle agglutination) |
| Detection Method | Microplate spectrophotometer read at 450 nm | Automated clinical chemistry analyzers read at 570 nm |

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

| STANDARDS |
| --- |
| Title and Reference Number |
| CLSI: Evaluation of the Linearity of Quantitative Measurement Procedures: A Statistical Approach; Approved Guideline (EP6-A). |
| CLSI: Interference Testing in Clinical Chemistry; Approved Guideline (EP 7-A) |
| CLSI: Method Comparison and Bias Estimation Using Patient Samples; Approved Guideline (EP09-A2) |
| CLSI: Evaluation of Precision Performance of Clinical Chemistry Devices; Approved Guideline (EP5-A) |

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CLSI: Protocols for Determination of Limits of Detection and Limits of Quantitation; Approved Guideline (EP9-A)

# L. Test Principle:

The diaDexus PLAC Test is based on turbidimetric immunoassay technology utilizing two $\mathrm{Lp - PLA_2}$ specific monoclonal antibodies (2C10 and 4B4) coated to latex microparticles. A set of $\mathrm{Lp - PLA_2}$ calibrators is used to plot a standard curve of absorbance (y-axis) versus $\mathrm{Lp - PLA_2}$ concentration in ng/mL (x-axis) from which the $\mathrm{Lp - PLA_2}$ concentration in the test sample can be determined. Two levels of controls are assayed to monitor performance within the clinical range of the assay. The concentration of $\mathrm{Lp - PLA_2}$ in each sample and control is interpolated from the standard curve using a spline curve fit with calibration curve fitting software.

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

The PLAC Test was validated on the Hitachi® 917.

# 1. Analytical performance:

# a. Precision/Reproducibility:

Intra-assay, inter-assay and total precision was assessed according to CLSI EP5-A2 with three reagent lots. Two controls (143.9 and $449.5\mathrm{ng / mL}$ ) and one serum $(68.5\mathrm{ng / mL})$ sample were run in duplicates in 2 separate runs on the Hitachi 917 analyzer for 20 days. The results shown below met the sponsors acceptance criteria (intra-assay precision $\mathrm{CV} < 8\%$ and total precision $\mathrm{CV} < 10\%$ ).

| Reagent Lot | Sample | Mean Lp-PLA2 Conc. (ng/mL) | Intra-assay %CV n=80 | Inter-assay %CV n=20 | Total %CV n=80 |
| --- | --- | --- | --- | --- | --- |
| 1 | 1 | 69 | 2.9 | 0.6 | 3.0 |
| | 2 | 143 | 2.4 | 1.3 | 2.7 |
| | 3 | 449 | 1.4 | 1.0 | 1.7 |
| 2 | 1 | 69 | 2.6 | 1.4 | 2.9 |
| | 2 | 143 | 1.8 | 1.3 | 2.2 |
| | 3 | 449 | 1.7 | 0.6 | 1.8 |
| 3 | 1 | 69 | 2.4 | 2.1 | 3.2 |
| | 2 | 144 | 2.0 | 1.6 | 2.5 |
| | 3 | 450 | 1.6 | 0.8 | 1.8 |

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b. Linearity/assay reportable range:

The recovery of reportable range was assessed according to CLSI EP6-A. Two high Lp-PA2 serum samples were mixed with low samples to product 11 concentrations/data points. The samples were assayed in duplicates with 3 lots on a single analyzer. The five level dilution series was conducted on a low serum sample. To further assess the lower end of the claimed assay range, the sponsor conducted an additional linearity study. Native human Lp-PLA2 was diluted with fetal bovine serum (used as a negative serum source) to the lowest level of detection. Percent recovery was determined and linear regression analysis was conducted. Additionally, the zero calibrator was titrated with an extremely high level of recombinant Lp-PLA2 up to 1500 ng/mL and dilutions were tested to assess the prozone or high dose hook effect. Three lots of reagents were evaluated over three separate runs on one analyzer. The results below met the sponsor's acceptance criterion (percent recovery between 90 and 110% of the established mean and no hook effect below 1500 ng/mL). The linear range is defined as 7-500 ng/mL.

| Reagent Lot | High Sample ng/mL | Low Sample ng/mL | Average % Recovery | Linear Regression Parameters | | |
| --- | --- | --- | --- | --- | --- | --- |
| | | | | Slope | Intercept | R2 |
| Human Serum (lot 1) | 483.4 | 109.9 | 94 | 1.00 | -13.6 | 0.995 |
| | 487.1 | 104.2 | 98 | 1.01 | -5.9 | 0.998 |
| Human Serum (lot 2) | 432.6 | 94.7 | 96 | 1.01 | -8.8 | 0.998 |
| | 461.8 | 89.7 | 99 | 1.01 | -5.1 | 0.999 |
| Human Serum (lot 3) | 460.2 | 98.3 | 95 | 1.03 | -15.8 | 0.994 |
| | 472.3 | 95.8 | 98 | 1.00 | -5.6 | 0.999 |
| Human Serum | 89.0 | 12.3 | 94 | 1.02 | -3.3 | 0.992 |
| Human High, bovine low | 438.8 | 0 | 100 | 1.04 | -6.9 | 0.997 |
| Human High, bovine low | 312.7 | 0 | 99 | 1.04 | -6.9 | 0.997 |
| Human High, bovine low | 213.6 | 0 | 95 | 1.04 | -6.9 | 0.997 |

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

The purified recombinant Lp-PLA2 antigen is utilized as the analyte in the

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PLAC test calibrators and controls. A master calibrator was prepared from purified primary stock solution of recombinant Lp-PLA2 that was quantitated by amino acid analysis. A master calibrator was prepared and the value was assigned using a panel of stored human serum samples that were characterized by multiple runs with the predicate device. The calibrators are manufactured to match the absorbance levels of the master calibrator at each calibration level (0, 50, 100, 250 and 500 ng/mL). Controls are manufactured to a target value, but assigned the actual value range generated by multiple PLAC test runs. Quality control acceptance criteria are that the calibrators must quantitate replicates of the quality control panel serum samples within +/- 10% of the established mean. The formulation used for both the calibrators and controls has been validated with the predicate device. The sponsor states a real-time stability of 16 months at 2 to 8°C.

d. Detection limit:

The sponsor conducted a Limit of Detection (LOD) and a Limit of Quantitation (LOQ) study. The detection limit was defined as the lowest concentration that is significantly different from zero. Calibrator 1 (0 ng/mL) and Calibrator 2 (20 ng/mL) were assay 20 times with three reagent lots in the same run. The mean of the zero calibrator plus 2 standard deviations was calculated as 4.5, 5.1 and 4.0 ng/mL. The limit of quantitation was defined as the lowest analyte concentration where the % CV of precision is less than 20%. The 50 ng/mL calibrator was titrated down to zero with calibrator diluent in a series of 10 incremental levels. Each sample was assayed in replicates of 3 in 10 runs by three reagent lots on one analyzer. The study was also performed with a serum sample diluted with bovine serum to 10 levels ranging from 56 ng/mL down to 8 ng/mL. The limit of quantitation was estimated to be 7 ng/mL in all three reagent lots using the calibrator series of samples and 11 ng/mL with the serum samples. The results from the above studies met the sponsor's acceptance criteria for LOD and LOQ of less than 20 ng/mL. The sponsor's limit of quantitation is estimated to be 7.0 ng/mL and limit of detection is 4.0 ng/mL.

e. Analytical specificity:

Five endogenous substances (hemoglobin, triglycerides, total cholesterol, bilirubin and human serum albumin) found in blood and exogenous substances (common and prescription drugs in the package insert) were evaluated for interference in the assay. Potential interfering substances were spiked with the interferent into four different serum samples with endogenous Lp-PLA₂ in the range of 160 to 470 ng/mL range. Defined amounts of the interferents at and exceeding a relevant physiological range were added to the serum samples, assayed in duplicate with three lots of reagents, and any resultant biases in analyte quantitation were reported. The highest levels were chosen according to CLSI Protocol EP7-A recommended high testing levels. Controls were prepared for each sample by spiking with the solvent used for

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each substance stock solution. Controls were matched to test samples for both volume and solvent concentration. Recoveries of test samples were calculated as result/matched control value x 100%. The sponsor's acceptance criterion was less than 10% error in detected Lp-PLA2 in the presence of the compound. There was no interference detected for any of the substances listed below.

| Endogenous | | Exogenous (OTC Drugs, etc.) | |
| --- | --- | --- | --- |
| Potential Interferent | Test Concentration | Potential Interferent | Test Concentration |
| Bilirubin | 20 mg/dL | Acetaminophen | 1.66 μmol/L |
| Cholesterol | 500 mg/dL | Aspirin | 3330 μmol/L |
| Hemoglobin | 10,000 mg/dL | Atorvastatin | 20 μmol/L |
| Triglycerides | 3000 mg/dL | Clopidogrel bisulfate | 100 μmol/L |
| Total Albumin* | ~6500 mg/dL | Diphenhydramine | 19.6 μmol/L |
| | | Fenofibrate | 125 μmol/L |
| | | Lisinopril | 0.74 μmol/L |
| * 2.5 g/dL albumin added to plasma pool of presumptively 4 g/dL albumin | Metformin | 310 μmol/L | |
| | | Niacin | 4800 μmol/L |
| | | Pravastatin | 100 μmol/L |
| | | Tolbutamide | 2400 μmol/L |
| | | Vitamin C | 227 μmol/L |
| | | Warfarin | 64.9 μmol/L |

f. Assay cut-off:

Not applicable.

2. Comparison studies:

a. Method comparison with predicate device:

The current PLAC Test turbidimetric immunoassay method was compared to the PLAC Test ELISA microplate assay (k062234). The singlet point method comparison for the 794 unaltered stored human serum samples ranged from <7 to 499 ng/mL, obtained from banked study sets were within the sponsor's linear range. The results are shown in the table below.

| Regression Parameters | | | | |
| --- | --- | --- | --- | --- |
| | Correl Coeff (r) | Slope | Intercept | N |
| Results | 0.92 | 1.02 | -24.5 | 794 |

b. Matrix comparison:

The sponsor conducted a recovery matrix and anticoagulant comparison study with the PLAC turbidimetric immunoassay. Ten plasma samples ranging from 79.4 to 201.3 ng/mL. The recovery ranges from 89 – 111% for EDTA

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plasma, 83-102% for Na heparin, 87- 106% for Li heparin. Specific recovery percentages linear equations for the 10 samples are shown in the table below.

| Sample | Serum | Plasma_K2 EDTA | %Rec Plasma to Serum | Na Heparin | %Rec Na Heparin to Serum | Lithium Heparin | %Rec Li Heparin to Serum |
| --- | --- | --- | --- | --- | --- | --- | --- |
| S01 | 180.0 | 182.8 | 102% | 187.0 | 104% | 188.0 | 104% |
| S02 | 112.9 | 101.7 | 90% | 92.5 | 82% | 103.8 | 92% |
| S03 | 165.6 | 170.3 | 103% | 182.3 | 110% | 180.7 | 109% |
| S04 | 155.3 | 173.5 | 112% | 152.0 | 98% | 163.1 | 105% |
| S05 | 91.9 | 95.5 | 104% | 79.4 | 86% | 82.7 | 90% |
| S06 | 197.4 | 201.3 | 102% | 204.9 | 104% | 211.5 | 107% |
| S07 | 70.3 | 79.4 | 113% | 80.7 | 115% | 81.7 | 116% |
| S08 | 157.1 | 156.6 | 100% | 162.6 | 103% | 166.4 | 106% |
| S09 | 161.2 | 142.2 | 88% | 149.5 | 93% | 140.8 | 87% |
| S10 | 170.3 | 166.5 | 98% | 172.2 | 101% | 176.9 | 104% |
| | | | 101% | | 100% | | 102% |
| | | | y=.977x +4.10 | | y=1.09x-13.641 | | y=1.08x-8.76 |
| | | | y=plasma x=serum | | y=Sodium Heparin x=serum | | y=Lithium Heparin x=serum |

Additionally, the sponsor conducted a matrix comparison study using 95 matched serum and EDTA samples that ranged from 132.1 to 402 ng/mL. The obtained recoveries ranged from 86.1 to 118.9% and the obtained linear regression equation was Y (serum) = 0.986(EDTA) +10.52.

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):

See clinical data provided in k030477.

4. Clinical cut-off:

Not applicable.

5. Expected values/Reference range:

The sponsor claims the same reference range as the initial device (k030477). The stored reference range samples were re-assayed by the PLAC test turbidimetric immunoassay to assess whether the results were statistically different from historical results. 155 plasma samples (61 females and 94 males) were assayed by the PLAC Test turbidimetric immunoassay in duplicate with one lot of reagents.

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Additionally, the sponsor tested the samples against the ELISA PLAC test k050523. The mean results for both tests were compared with the historical reference results using the Two Sample t-Test for assessment of statistical differences. The sponsor's acceptance criterion is a Two-Sample t-Test analysis result of $p \geq 0.05$ , which states no statistical difference. The results are presented in the table below.

| | Lp-PLA2 ng/mL | | |
| --- | --- | --- | --- |
| Method | k072599 Turbidimetric immunoassay | k050523 ELISA | k030477 Historical Values |
| Lp-PLA2 Percentile | | | |
| 5 | 172 | 195 | 157 |
| 25 | 212 | 229.7 | 201.7 |
| 50 (Median) | 245.4 | 261 | 234.9 |
| 75 | 295 | 294 | 274 |
| 95 | 359 | 358 | 349 |
| Mean | 253 | 265 | 241 |
| 95% CI of Mean | 244–263 | 256–273 | 232–250 |
| N | 155 | 155 | 155 |
| P value turbidimetric vs. historical | 0.0842 | | |
| P value turbidimetric vs. ELISA | 0.0539 | | |

The sponsor has chosen to use the previously obtained expected values from k030477 that are stated below.

" Samples from apparently healthy males (n=251) and apparently healthy females (n=174), in the clinically relevant age range of 40 to 70 years, were evaluated with the diaDexus PLAC Test. The reference population was represented by the following ethnic backgrounds: African-American n=26, Caucasian n=390, Hispanic n=8 and not specified n=1. The distributions of Lp-PLA $_2$ values across the entire population and divided by gender appear in the following table:

| | Lp-PLA2 ng/mL | | |
| --- | --- | --- | --- |
| Percentile | All (n=425) | Females (n=174) | Males (n=251) |
| 5 | 126 | 120 | 131 |
| 20 | 174 | 169 | 179 |
| 33 | 201 | 188 | 205 |
| 50 | 235 | 228 | 244 |
| 67 | 262 | 252 | 268 |
| 80 | 289 | 285 | 293 |
| 95 | 369 | 342 | 376 |

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The reference interval calculated from the samples (central 90%) was found to be 120–342 ng/mL for females and 131–376 ng/mL for males."

**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.

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