Intended Use and Method

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.

Summary

Lp-PLA2 is a calcium-independent serine lipase that is associated with low-density lipoprotein (LDL) in human plasma and serum and is distinct from other phospholipases such as cPLA2 and sPLA2. Lp-PLA2 is produced by macrophages and is expressed in greater concentrations in atherosclerotic lesions. Several lines of evidence suggest that oxidation of LDL plays a critical step in the development and progression of atherosclerosis. Lp-PLA2 participates in the oxidative modification of LDL by hydrolyzing oxidized phosphatidylcholines, generating lysophosphatidylcholine and oxidized free fatty acids, both of which are potent pro-inflammatory products that contribute to the formation of atherosclerotic plaques. The PLAC test does not replace blood cholesterol tests or other traditional risk factors identified for coronary heart disease or ischemic stroke.

Test Kit Reagents and Materials

(Sufficient for 96 wells)

Antibody-coated strips - 12 each x 8 microwell strips with frame
Mouse monoclonal anti-Lp-PLA2 (2C10) antibody-coated microwell strips
Calibrators 1-6 - 1 set, Liquid
(0, 50, 100, 250, 500, and 1000 ng/mL)
diaDexus recombinant Lp-PLA2 antigen (DDX-RA) in a protein-stabilizing diluent
Assay buffer - 23 mL, Blue
Buffered reagent with bovine serum albumin and mouse serum
Conjugate - 23 mL, Red
Mouse monoclonal anti-Lp-PLA2 (4B4) antibody conjugated to horseradish peroxidase in a buffered reagent with carrier proteins (fetal calf serum and mouse IgG)
TMB reagent - 11 mL
3,3',5,5'-tetramethylbenzidine in a mildly acidic buffer
Stop solution - 11 mL
1N HCl

Reagent preparation and storage

Unopened test kits should be stored at 2-8°C (35.6-46.4°F) upon receipt. In addition, the microwell strips should be kept sealed in the foil pouch with desiccant to minimize exposure to moisture. Opened kits will remain stable until the expiration date shown, provided they are stored as described above.

Expected values
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

Reference interval calculated from blood samples of apparently healthy males (n=251) and females (n =174) in the clinically relevant age range of 40-70 years. The reference population was represented by various ethnic backgrounds, including African-American, Caucasian, and Hispanic. These ranges are provided only as guidelines. Each laboratory should establish its own reference intervals.

Sensitivity

The minimum detection limit, as calculated by interpolation of the mean plus two standard deviations of 24 replicates of the 0 ng/mL Lp-PLA2 calibrator, is 1.3 ng/mL.

Assay precision

Intra-assay and inter-assay variability were determined by testing three human plasma pools with Lp-PLA2 concentrations distributed throughout the calibration range of the assay. The three plasma pools were assayed, using a single lot of reagents, in duplicate, on two separate stripwells per day, for twenty days.

Plasma pool
Mean concentration
Lp-PLA2 (ng/mL)
Intra-assay % CV
n=80
Inter-assay % CV
n=80
1
208
4.3
6.3
2
494
5.8
8.7
3
770
5.0
8.5

Interfering Substances
Five endogenous substances found in blood were evaluated for interference in the assay. No appreciable interference was observed at spiked levels of:

  • 500 mg/dL hemoglobin
  • 3000 mg/dL triglycerides
  • 500 mg/dL cholesterol
  • 20 mg/dL bilirubin
  • total of ~6 g/dL albumin

Six exogenous substances were evaluated at concentrations exceeding therapeutic blood levels and were determined not to interfere with the measurement of Lp-PLA2.

  • Acetylsalicylic Acid (Aspirin) 50 mg/dL
  • Acetaminophen (Tylenol®) 20 mg/dL
  • Ascorbic Acid (Vitamin C) 50 mg/dL
  • Diphenhydramine (Benadryl®) 50 mg/dL
  • Tolbutamide (Orinase®) 100 mg/dL
  • Pravastatin (Pravachol®) 446 µg/dL (10 µM)