10-0141 10-0145

10-0142 10-0146
Test for Lp PLA 2 10-0143 10-0147
10-0144 10-0148

PLAC® Test for Lp-PLA2 Activity

Enzyme Assay for the Quantitative Determination of Lp-PLA2 Activity
in Human Plasma or Serum

diaDexus, Inc.
349 Oyster Point Blvd.
South San Francisco, CA 94080 USA
Tel: 1-877-PLACTEST (1-877-752-2837)
Fax: 1-650-246-6499
www.plactest.com\

Symbol Key

Catalog Number Buffer

In Vitro Diagnostic Medical Device Substrate

Batch Calibrator
Expiration Date Control Low
8°C

2°C Store at 2-8 deg C Control High

Irritant Consult Instructions for Use
CAUTION: Federal law restricts the device
Manufacturer
to sale or on the order of a physician
Read

Read this package insert completely before using the product. Follow instructions carefully when performing tests. Failure
to follow the instructions may result in inaccurate results.

For specific automated clinical chemistry Analyzer Application Sheets, contact diaDexus Customer Service at 1-877-752-
2837.

This product is covered by U.S. Patent www.diadexus.com/legal/patents.php. Additional patents pending.

and PLAC are registered trademarks of diaDexus.

All other trademarks are property of their respective owners.
Copyright 2014 diaDexus, Inc. All rights reserved

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INTENDED USE
The PLAC® Test for Lp-PLA2 Activity is an enzyme assay for the in vitro quantitative determination of Lp-PLA2 (lipoprotein-
associated phospholipase A2) activity in EDTA plasma and serum on automated clinical chemistry analyzers. Lp-PLA2 ac-
tivity is to be used in conjunction with clinical evaluation and patient risk assessment as an aid in predicting risk of coronary
heart disease (CHD) in patients with no prior history of cardiovascular events.

SUMMARY AND EXPLANATION

Lp-PLA2 is a calcium-independent phospholipase A2 enzyme that is associated with both low-density lipoprotein (LDL)
and, to a lesser extent, high-density lipoprotein (HDL) in human plasma and serum (Zalewski and MacPhee, 2005) and is
distinct from other such phospholipases such as cPLA2 and sPLA2 (Kudo and Murakami 2002, Burke and Dennis 2009).
Lp-PLA2 is produced by macrophages and other inflammatory cells and is expressed in greater concentrations in advanced
atherosclerotic lesions than early-stage lesions (Hakkinen, Luoma et al. 1999, Kolodgie, Burke et al. 2006). Several lines
of evidence suggest that oxidation of LDL plays a critical step in the development and progression of atherosclerosis (Wit-
ztum 1994, Chisolm and Steinberg 2000). Lp-PLA2 participates in the breakdown of oxidized LDL in the vascular wall by
hydrolyzing the oxidized phospholipid, producing lysophosphatidylcholine and oxidized free fatty acids, both of which are
potent pro-inflammatory products that contribute to the formation of atherosclerotic plaques (Macphee, Moores et al. 1999,
Macphee 2001, Suckling and Macphee 2002). Lp-PLA2 has demonstrated modest intra- and inter-individual variation, com-
mensurate with other cardiovascular lipid markers and substantially less variability than high sensitivity C-reactive protein
(hs-CRP). In addition, Lp-PLA2 is not elevated in systemic inflammatory conditions, and may be a more specific marker of
vascular inflammation. The relatively small biological variation of Lp-PLA2 and its vascular specificity are of value in the
detection and monitoring of cardiovascular risk (Wolfert, Kim et al. 2004, Lerman and McConnell 2008, Thompson, Gao et
al. 2010).

PRINCIPLE OF THE TEST

The PLAC Test for Lp-PLA2 Activity is an enzyme assay. Lp-PLA2, in plasma or serum, hydrolyzes the sn-2 position of the
substrate, 1-myristoyl-2-(4-nitrophenylsuccinyl) phosphatidylcholine, producing a colored reaction product, 4-nitrophenol.
The rate of formation of 4-nitrophenol is measured spectrophotometrically and the Lp-PLA2 activity is calculated from the
rate of change in absorbance. A set of five Lp-PLA2 calibrators is used to generate a standard curve fit of change in absor-
bance versus Lp-PLA2 activity level in nmol/min/mL from which the sample Lp-PLA2 activity is derived.

REAGENTS AND MATERIALS

The PLAC Test for Lp-PLA2 Activity is supplied with:
• Lp-PLA2 Substrate, 1-myristoyl-2-(4-nitrophenylsuccinyl) phosphatidylcholine
• Calibrators
• Control Low
• Control High
• Buffer
• Certificate of Analysis – Control Range
Other materials required but not provided:
• Automated Clinical Chemistry Analyzer and System Operation Manual.
• Analyzer Application Sheet specific to the clinical chemistry analyzer used is available separately. Contact diaDexus
Customer Service.

WARNINGS AND PRECAUTIONS

Dilution of samples will create erroneous results. Samples cannot be diluted at any Lp-PLA2 activity level.
Samples with Lp-PLA2 values above the measuring range should be reported as >382 nmol/min/mL. In the Clinical
Validation Study, > 99% of subjects reported values ≤382 nmol/min/mL.

Hemolyzed samples interfere with the assay and should not be tested.
Samples that are visibly hemolyzed should be redrawn. Testing hemolyzed samples with >1.0 mg/mL hemoglobin may
cause erroneous results.

Reversing the positions of the reagents on the analyzer will lead to erroneous results.
Make sure to load the R1 reagent in the right location on the analyzer, R1 position, and the R2 reagent in the instrument’s
R2 position.

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 • For In Vitro Diagnostic Use.
• CAUTION: Federal law restricts the device to sale by or on the order of a physician.
• Treat all blood samples, calibrators and controls as potentially biohazardous material.
• Visually observe samples for excessive turbidity and clots prior to analysis. Samples with excessive turbidity and clots
may impact results and should not be used.
• Dispose of reagents in a manner consistent with relevant regulations.
• Do not use reagents, calibrators or controls past their expiration dates.
• Do not mix reagents, calibrators or controls from different kit lots.

REAGENT PREPARATION AND STORAGE

Reagents are provided ready to use. Remove the caps of reagent R1 and R2 and place on the instrument. Reagents are
stable for up to 4 weeks on board the analyzer.
Once opened:
• Reagents can be stored at 2-8°C for up to 4 weeks
• Calibrators can be stored at 2-8°C for up to 3 months
• Controls can be stored at 2-8°C for up to 3 months
Please refer to the specific clinical chemistry Analyzer Application Sheet for information specific to your analyzer. Laborato-
ries should verify on board reagent stability on their own analyzers under typical laboratory conditions.

SPECIMEN COLLECTION AND STORAGE

• Fasting is not required.
• Collect whole blood by venipuncture in:
-- K2 EDTA plasma collection tubes with or without gel
-- K3 EDTA plasma collection tubes without gel
-- Serum collection tubes, with or without gel.

• Process blood using standard separation procedures.

-- Whole blood can be kept up to 4 hours at 20-22°C or up to 30 hours at 2-8°C prior to separation.
• Following centrifugation:
-- Examine for hemolysis. If present, discard sample and re-draw.
• Sample can be tested immediately or stored prior to testing under the following conditions:
-- 24 hours at 20-26°C
-- Up to 2 weeks at 2-8°C
-- Up to 18 months at -20°C
-- Up to 2 years at -70°C
-- Plasma and serum samples can be freeze-thawed up to 5 times after freezing at either -70°C or -20°C.
-- When transporting samples, ship samples on cold packs at 2-8°C.
• Handle and dispose of all samples using Universal Biohazardous Precautions.

ASSAY PROCEDURE

Calibration
The assay is calibrated using a 5-point calibration curve. A standard curve is generated using the appropriate curve fit model
indicated in the Analyzer Application Sheet. Verify the calibration with at least two levels of controls according to the labora-
tory’s requirements. Recalibrate and run controls for each kit from a new lot and thereafter, every 4 weeks for kits from the
same lot. If controls fall outside of laboratory’s acceptable range, recalibrate as necessary up to the expiration date of the
opened reagents.

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Quality Control
Test at least two levels of an appropriate quality control material a minimum of once per day for each day of use. In addition,
run controls after each new calibration run. It is recommended that low and high controls be included in each run. If control
values are not within acceptance limits, repeat the assay. Additional quality control testing may be necessary according to
local, state and/or federal regulations or accreditation requirements.

Example Assay Procedure

The PLAC Test for Lp-PLA2 Activity should be run using the appropriate settings for the analyzer to be used. For detailed
instructions and settings for each analyzer, please refer to the Analyzer Application Sheet for the specific automated clini-
cal chemistry analyzer used. A general explanation of the assay procedure for the Beckman Coulter (Olympus) AU400®
Analyzer is described.

Settings for the Beckman Coulter (Olympus) AU400 Clinical Chemistry Analyzer
Assay Code Rate
Assay Time 8.5 minutes
Read Cycle 12 to 14
Sample Volume 25 µL
Reagent R1 vol. 100 µL R1 reagent (R1 position)
Reagent R2 vol. 25 µL R2 reagent (R2 position)
Wavelength 1° 410 nm, 2° 520 nm
Calibration Method Spline 5 point
Assay Range 10 to 382 nmol/min/mL

Procedural Notes
• It is recommended that each lab determine a suitable calibration frequency. At a minimum, a new calibration curve
should be generated with a kit from a new lot, and thereafter, every 4 weeks for kits from the same lot. Run calibration
when and if controls fall outside acceptable range.
• Upon storage, do not switch caps on reagent solutions as this may lead to contamination.
• All samples should be well mixed before testing, and especially after thawing stored samples. A vortex mixer may be
used; however, any air bubbles or foaming of the samples should be avoided.

Example of Calibration Curve

Lp-PLA2 Activity Absorbance

nmol/min/mL OD/min
0 0.0067
50 0.0857
100 0.1601
250 0.3469
400 0.5142

LIMITATIONS
• Reliable, accurate and reproducible results will be obtained when the assay procedure is performed with a complete
understanding of the package insert instructions and with adherence to good laboratory practice.
• As with any analytical method, the possibility exists that substances and/or factors not tested (e.g. technical or proce-
dural) may interfere with the test and cause false results. Results should be considered in conjunction with other clinical
and analytical methods.
Interpretation of Results
The PLAC Test for Lp-PLA2 Activity provides information on the risk of coronary heart disease (CHD) events. In the Clinical
Validation Study (see below), the overall population rate of CHD events at 5-year follow-up was 4.1% (95% CI 3.8 - 4.38%),

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prior to consideration of subject PLAC Activity values. Using a PLAC Activity cut-point value of 225 nmol/min/mL, subjects
were then classified into a high PLAC Activity group (Lp-PLA2 activity ≥ 225 nmol/min/mL) or a low PLAC Activity group (Lp-
PLA2 activity < 225 nmol/min/mL). The following absolute rates of CHD events at 5-year follow-up were observed (including
95% CI):

• High PLAC Activity group CHD event rate 7.0% [6.2% - 7.8%]
• Low PLAC Activity group CHD event rate 3.3% [3.0% - 3.6%]
The high PLAC Activity group was found to have a 5-year absolute rate for CHD events that was over twice that of the low
PLAC Activity group. The difference in absolute rates between the two PLAC Activity groups is both statistically significant
(p <0.001) and clinically important given the continuing lifetime risk of CHD events. The finding that an individual with no
prior history of cardiovascular events has high PLAC Activity would suggest that they are at higher than average risk. Simi-
larly, the finding that such an individual has low PLAC Activity would suggest that they are at lower than average risk.

Additional analyses showed that high PLAC Activity was a significant predictor of CHD events relative to low PLAC Activity,
even after adjustment for other demographic and cardiovascular risk factors including age, race, gender, smoking, hyperten-
sion, diabetes, LDL-cholesterol and HDL-cholesterol.

Use of PLAC Activity results in conjunction with the other clinical parameters (e.g., age, life expectancy, etc.) and traditional
risk factors (e.g., LDL-cholesterol, total cholesterol, blood pressure, etc.) may be used to refine risk assessment in primary
prevention.

Cut-Point
Analyte Units Reduced Risk Increased Risk
Lp-PLA2 Activity nmol/min/mL < 225 ≥ 225

PERFORMANCE CHARACTERISTICS
Performance characteristics were established using one Beckman Coulter (Olympus) AU400 Analyzer. Refer to the specific
clinical chemistry Analyzer Application Sheet for performance characteristics.

Analytical Sensitivity
The analytical sensitivity (limit of quantitation) of the assay is 10 nmol/min/mL.

Assay Precision
Within-run and within-lab variability were determined by testing four human plasma samples and two controls with Lp-PLA2
activities ranging from 113 to 315 nmol/min/mL. Samples were assayed in duplicate, twice a day, over 20 days and with 3 kit
lots using one instrument. Total precision CV’s for each reagent lot and sample were <3%. The assay results for within-run
repeatability and total lab variability which includes within-run, between-run, between-day and between-lot variance compo-
nents are summarized below for each sample tested:
Mean Value Repeatability (within-run) Within-Lab Precision
Sample (nmol/min/mL)
n=240 SD %CV SD %CV

Kit Control Low 120.0 1.81 1.5% 3.40 2.8%

Kit Control High 302.8 4.31 1.4% 8.24 2.7%
Plasma 1 113.0 1.41 1.2% 4.24 3.8%
Plasma 2 208.1 2.97 1.4% 6.94 3.3%
Plasma 3 244.4 3.67 1.5% 8.61 3.5%
Plasma 4 314.6 3.86 1.2% 9.79 3.1%

Linearity
Several dilution series were prepared from plasma samples with known high and low Lp-PLA2 activity levels and were tested
with 3 kit lots. In the dynamic range of 6 to 382 nmol/min/mL, linear regression of Lp-PLA2 activity levels resulted in slopes
ranging from 0.98 to 1.04, with intercepts ranging from -0.40 to -0.03 nmol/min/mL and R2 values ranging from 0.995 to
0.999. Linearity was demonstrated from 10 to 382 nmol/min/mL with a deviation from linearity of ≤ 10%. The measuring
range of the assay is determined to be 10 to 382 nmol/min/mL.

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Interfering Substances
Endogenous substances were titrated into samples (above and below the Lp-PLA2 cut-point) with known levels of each
endogenous substance and were tested. No appreciable interference was observed for the following substances:

Interference Testing: Endogenous Substances

Potential Interferent High Concentration
Albumin, g/L 60
Unconjugated Bilirubin, mg/dL 20
Conjugated Bilirubin, mg/dL 12
Cholesterol, mg/dL 300
Triglycerides, mg/dL 400
Hemoglobin, mg/mL 1

Exogenous substances (common and prescription drugs) were evaluated for interference in the assay. Samples were
spiked with two levels of the potential interferent and tested. No appreciable interference was observed for the following
substances at the spiked levels tested.

Interference Testing: Exogenous Substances

Potential Interferent Test Concentration Low Test Concentration High

Acetaminophen, µmol/L 33 1324

Aspirin, µmol/L 720 3600
Atorvastatin, µmol/L 2 20
Diphenhydramine, µmol/L 2 20
Fenofibrate, µmol/L 42 125
Lisinopril, µmol/L 0.25 0.74
Niacin, µmol/L 480 4800
Tolbutamide, µmol/L 400 2300
Warfarin, µmol/L 10 33
Metformin, µmol/L 31 310
Clopidogrel bisulfate,
10 100
µmol/L
Vitamin C, µmol/L 14 342

Recovery
Various amounts of a high Lp-PLA2 activity level solution were added to an enzyme-free diluent to create seven activity
levels. These spiked solutions were assayed with 3 lots of reagents and the Lp-PLA2 activity levels were then compared to
expected values resulting in slopes ranging from 0.99 to 1.10, with intercepts ranging from -2.9 to 4.2 nmol/min/mL and R2
ranging from 0.997 to 1.000.

Clinical Studies
The clinical effectiveness of the PLAC Test for Lp-PLA2 Activity in its intended use population was validated in a Clinical Vali-
dation Study utilizing participants in the REasons for Geographic And Racial Differences in Stroke (REGARDS) study. Be-
gun in 2003, REGARDS is an active observational population study which continues to follow its participants longitudinally
for the development of cardiovascular events (CHD and Stroke). Designed to elucidate racial and geographical differences
in cardiovascular disease within the general US population, the study targeted a balanced enrollment of gender and race,
and enrolled Black and White participants only. The REGARDS study design, inclusion and exclusion criteria, and coronary
heart disease outcomes have been previously published (Howard, Cushman et al. 2005, Safford, Brown et al. 2012). With
its nationwide scope and 30,183 total enrolled participants, REGARDS is one of the largest single such study ever under-
taken in the United States by the National Institutes of Health.

The primary endpoint used in the Clinical Validation Study for the PLAC Test for Lp-PLA2 Activity was a composite of total
CHD events, comprising 1) acute myocardial infarction, 2) coronary revascularization, and 3) CHD-related death.

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The Clinical Validation Study used a case-cohort study design (Prentice 1986). Consistent with the intended use, subjects
were randomly sampled from all REGARDS participants with no prior history of cardiovascular events at baseline enrollment
(23,019 participants). This sampled population was then enriched with all remaining REGARDS CHD cases that occurred
after the same baseline exclusion.

The resulting Clinical Validation Study is a case-cohort of 4,598 subjects, including a total of 933 cases and 3,665 controls.
In the sampled population, subject ages ranged from 45 to 92 years (median 63 years), with gender distributed 41.7% male
and 58.3% female, and race distributed 41.5% black and 58.5% white. Statistical tests confirmed that the case-cohort was
representative of the full REGARDS population for the intended use. Efficacy analyses, Kaplan-Meier and Cox proportional
hazards models were all weighted to adjust the Clinical Validation Study to the underlying prevalence of cases in the parent
study population (Barlow 1994).

Frozen EDTA plasma specimens were tested with the PLAC Test for Lp-PLA2 Activity following the recommended sample
handling protocol and the observed values were analyzed. The median population follow-up was 5.3 years from the draw
date of the specimens tested.

PLAC Activity Distribution: PLAC Test for Lp-PLA2 Activity values in the sampled population ranged from a minimum of
8 to a maximum of >382 nmol/min/mL, with a median value of 178 nmol/min/mL (interquartile range (IQR) 145 – 216 nmol/
min/mL).

Analysis Cut-point: For the Clinical Validation Study efficacy analyses, a PLAC Test for Lp-PLA2 Activity cut-point of 225
nmol/min/mL was pre-specified based on prior studies and publications using PLAC activity in other independent cohorts.
This analysis cut-point was used as a binary classifier in the study, dividing the study population into the low (below the cut-
point) and high (at or above the cut-point) PLAC Activity groups.

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Kaplan-Meier Analyses: In the Kaplan-Meier analysis of the Clinical Validation Study, absolute risk of CHD events was
higher in the high PLAC Activity group (log-rank p-value < 0.001), as seen in the figure below. The absolute risk of CHD
events of the two groups separated early and was consistently different after one year of follow-up and at all subsequent
timepoints pre-specified for the analysis. The same analysis was also statistically significant within each gender and race
analyzed independently (log-rank p-value < 0.001 for each analysis).

Absolute Rates of CHD by PLAC Activity Group: The absolute risk of CHD events at 5-year follow-up is presented for
each PLAC Activity group of the Clinical Validation Study in the table below. The study indicated that the absolute risk of
CHD events in the high PLAC Activity group is 2.1 times the absolute risk of CHD events in the low PLAC Activity group.

5-Year Risk of CHD Events (Kaplan-Meier Absolute Risk/Rate Analysis)

Absolute Risk Absolute Risk
Absolute Risk Post-Test Post-Test Relative Risk
Population
Pre-Test Low Group High Group (High/Low)
(<225 nmol/min/mL) (≥225 nmol/min/mL)
All 4.1% 3.3% 7.0% 2.1

Females 2.7% 2.4% 5.3% 2.2

Males 6.1% 5.2% 7.7% 1.5

White 4.4% 3.5% 6.9% 2.0

Black 3.6% 3.2% 7.6% 2.4

Race by Gender Subgroup Analyses: The predictive power of the PLAC Test for Lp-PLA2 Activity was also assessed for
each race by gender subgroup of the Clinical Validation Study. The figure below shows the predictive power of PLAC Activ-
ity within each race by gender subgroup using univariate Cox proportional hazards model, [high versus low PLAC activity
group] with 5-year Kaplan-Meier rate estimates provided for each PLAC Activity group. The PLAC Test for Lp-PLA2 Activity
was shown to be a statistically significant predictor of total CHD events in all subgroups, with a consistently higher hazard
ratio (HR) estimate observed for women compared to that for men, regardless of race.

8
5-Year Risk of CHD Events and Cox Proportional Hazards Models by Race by Gender Subgroup

Kaplan-Meier Analyses in Each Race by Gender Subgroup

9
Risk Adjusted Cox Proportional Hazards Models: Cox proportional hazards models showed the PLAC Test for Lp-PLA2
Activity to be a statistically significant predictor of total CHD events when comparing the high versus the low PLAC Activity
groups of the Clinical Validation Study, with a fully-adjusted hazard ratio of 1.54 (1.31 – 1.82, p < 0.001) after adjustment for
age, gender, race, diabetes, hypertension, smoking, LDL and HDL.

Secondary Component Endpoint Analysis: The predictive power of the PLAC Test for Lp-PLA2 Activity was also assessed
using Cox proportional hazards models for each of the individual component endpoints of the Clinical Validation Study. The
PLAC Test for Lp-PLA2 Activity was shown to be a consistent and statistically significant predictor for each of acute myocar-
dial infarction, coronary revascularization and CHD-related death (p < 0.001 for each analysis).

Expected Values
EDTA-plasma samples were obtained from an all-comers population of 300 subjects of a clinically relevant age of 35 to 75
years old (median age 57) from two different geographical locations. Information regarding age, gender and race was col-
lected from each subject. No health information was obtained. The testing population included 154 males and 146 females
with a racial distribution of 38% White, 32% Black, 21% Hispanic and 9% Asian. Samples were tested with the PLAC Test
for Lp-PLA2 Activity following the recommended sample handling protocol and the expected values were computed.
The distribution of baseline PLAC activity values for this population are shown in the following table:
All (N=300) Female (N=146) Male (N=154)
Percentile Lp-PLA2 Lp-PLA2 Lp-PLA2
(nmol/min/mL) (nmol/min/mL) (nmol/min/mL)
Min 50 50 70
2.5 84 74 92
5 94 86 102
25 137 130 149
33 148 139 155
50 167 154 176
67 196 179 204
75 211 200 219
95 276 264 295
97.5 303 300 329
99 369 339 >382
Max >382 370 >382
Mean 176 166 186

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A low-frequency homozygous null mutation (V279F) has been reported in individuals with two Asian parents that results in
a low level of circulating Lp-PLA2 (Jang, Waterworth et al. 2011). Such individuals generally present with Lp-PLA2 activity
values that are either unmeasurable or below the minimum population values shown in the previous table.

These reference ranges are provided as guidelines only and are not intended to address “critical values” or medical deci-
sion limits. The results indicate that each clinical laboratory may observe a median and range of Lp-PLA2 activity values that
depend on the patient population tested (e.g. patients that are generally at greater risk for cardiovascular disease versus
those at a generally lower risk). Each laboratory should establish its own reference intervals.

The distribution of PLAC Activity values observed in the Clinical Validation Study, which enrolled Blacks and Whites, en-
compasses those seen in other races (tested in a separate study) as shown in the figure below for Asians (n = 285) and
Hispanics (n = 199).

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PRODUCT SAFETY INFORMATION
Calibrator Set (1-5), Control Low and High R1 Buffer
R36 R36
S25/26/36/37/39 S25/26/36/37/39

R36 Irritating to eyes

S25 Avoid contact with eyes
In case of contact with eyes, rinse immediately with plenty of water and seek
S26
medical advice
S36/37/39 Wear suitable protective clothing, gloves, eye/face protection

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Hofman, C. Packard, S. G. Thompson, R. Collins and J. Danesh (2010). “Lipoprotein-associated phospholipase A(2) and risk of coronary
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associated phospholipase A2 (Lp-PLA2), a novel marker of cardiovascular risk (abstract).” Circulation 110(Supplement 3): 309.
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P/N 30050-01
2015-01-15
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