Reproducibility, Pre-Analytical Variables and Effect of Aspirin On Soluble P Selectin and CD40 Ligand.

Several studies have demonstrated a significant association between platelet plasma markers (soluble CD40 ligand [sCD40L] and soluble p selectin [sPselectin]) and in-vivo platelet activation and cardiovascular events. However, the reliability and pre-analytical variation of these assays remain uncertain. We therefore sought to investigate the reproducibility of these assays, type of collection (plasma or sera), correlation between sCD40L and sPselectin, and the effect of aspirin.

Following an overnight fast, 40 healthy volunteers had early morning phlebotomy during 4 consecutive weeks. Subjects took aspirin 81mg daily x7 days between weeks 3 and 4. Platelet poor plasma was obtained via centrifugation at 2500 × g for ten minutes within 15 minutes of phlebotomy while serum samples were similarly processed at 30 minutes after phlebotomy. Samples were aliquoted and stored at −80°C no more than five minutes after completion of centrifugation. Concentrations of sPselectin and sCD40L were determined in plasma and serum by quantitative enzyme linked immunosorbent assay (Bender MedSystems: BMS219 & BMS293, respectively). Reproducibility over time of levels of sPselectin and sCD40L was assessed by coefficient of variation (CV). Correlation was assessed using Pearson r statistic. The difference between levels pre and post aspirin was measured with Wilcoxon Signed- Rank test. Data is presented as median [interquartile range].

Soluble CD40L measurements were reproducible over time in plasma (Week 1: 0.72 ng/mL [0.35–1.63], Week 2: 0.72 [0.36–1.69], Week 3: 0.66 [0.34– 1.7]; CV: 9.6 %) and serum (Week 1: 2.43 [2.12– 2.99], Week 2: 2.26 [1.8– 2.98], Week 3: 2.44 [1.75– 3.05]; CV 8%). Soluble P-selectin measurements were also reproducible over time in plasma (Week 1: 58.9 ng/mL [46.2–70.8], Week 2: 55.5 [45.4–68.6], Week 3: 52.6 [43.8–62.8]; CV: 9.4%) and serum (Week 1: 116 ng/mL [94.6–145.5], Week 2: 113.6 [90.7–149.6], Week 3: 111.9 [94.1–148.8]; CV: 6%). Measurement of sCD40L in plasma correlated with levels in serum before aspirin (r=0.88, p< 0.0001) and after aspirin (r=0.87, p< 0.0001) as did levels of sPselectin in plasma correlate with levels in serum before aspirin (r=0.66, p< 0.0001) and after aspirin (r=0.54, p= 0.0004). There was no significant correlation between sCD40L and sPselectin before (r=-0.06, p=0.704) or after aspirin (r=-0.0956, p=0.573) in plasma or in sera (before aspirin, (r= 0.03, p=0.853) or after aspirin (r=0.11, p=0.482)). After 1-week of aspirin 81mg/day, there was a reduction in sCD40L in serum (2.43 ng/mL vs. 2.07; p<0.0001) and plasma (0.72 ng/mL vs. 0.64; p=0.245), however, the latter was not statistically significant. There was a significant reduction in sPselectin following a week of low-dose aspirin in both serum (116 ng/mL vs. 107.1; p<.0001) and plasma (58.9 ng/mL vs. 51; p=0.019).

Soluble CD40L and sPselectin are independent markers that are reproducible in both plasma and sera (CV <10% for every comparison) when performed in a laboratory with standardized methodology. One week of low-dose aspirin (81mg/day) effectively reduces sPselectin and sCD40L.

No relevant conflicts of interest to declare.