title: Influence of pre-analytical and analytical factors on soluble CD40L measurements.
abstract: The soluble form of CD40L (CD40 ligand), a pro-atherogenic mediator, has emerged as a diagnostic and prognostic marker for cardiovascular events. However, as platelets can shed CD40L upon activation, accurate measurement has proved challenging. The present study addresses the controversy regarding the appropriate specimen and preparation for laboratory evaluation of blood sCD40L (soluble CD40L). Serum and plasma (collected in EDTA, citrate or heparin) were collected from healthy volunteers (n=20), and sCD40L was analysed by ELISA immediately or after one to three freeze-thaw cycles and at different centrifugation speeds. Urine sCD40L levels were measured in subjects with low- and high-plasma sCD40L levels. Serum sCD40L levels (5.45+/-4.55 ng/ml; P<0.001) were higher than in citrate, EDTA or heparin plasma (1.03+/-1.07, 1.43+/-1.03 or 1.80+/-1.25 ng/ml respectively), with no significant differences between plasma preparations. Increasing g values (200-13000 g), which gradually deplete plasma of platelets, yielded lower sCD40L levels. Repeated freeze-thaw cycles significantly (P<0.05) increased sCD40L concentrations in platelet-rich, but not platelet-depleted, plasma (up to 2.4-fold). Bilirubin and haemoglobin interfered positively, and triacylglycerols (triglycerides) and cholesterol quenched CD40L signalling. No sCD40L was detected in urine samples. In conclusion, serum yields higher sCD40L concentrations than plasma; accurate measurements of sCD40L require exclusion of platelets and avoiding their post-hoc activation. Samples with high concentrations of bilirubin, haemoglobin and/or triacylglycerols should be excluded, as these substances interfere with the assay.
authors.affiliation: Donald W. Reynolds Cardiovascular Clinical Research Center, Department of Medicine, Brigham and Women's Hospital and Harvard Medical School, 77 Avenue Louis Pasteur, NRB 741, Boston, MA 02115, USA.
authors.forename: Nerea
authors.initials: N
authors.lastname: Varo
authors.forename: Rebecca
authors.initials: R
authors.lastname: Nuzzo
authors.forename: Cristina
authors.initials: C
authors.lastname: Natal
authors.forename: Peter
authors.initials: P
authors.lastname: Libby
authors.forename: Uwe
authors.initials: U
authors.lastname: Schönbeck
mesh_terms: D000328:Adult; D001663:Bilirubin / Q000494:pharmacology; D015415:Biomarkers / Q000097:blood; D001792:Blood Platelets / Q000737:chemistry; D001793:Blood Preservation / Q000379:methods; D001800:Blood Specimen Collection / Q000379:methods*; D023201:CD40 Ligand / Q000097:blood* / Q000187:drug effects / Q000652:urine; D019343:Citric Acid; D015925:Cryopreservation; D004492:Edetic Acid; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods; D006454:Hemoglobins / Q000494:pharmacology; D006493:Heparin; D006801:Humans; D008055:Lipids / Q000494:pharmacology; D008875:Middle Aged; D010949:Plasma / Q000737:chemistry; D044967:Serum / Q000737:chemistry; D012995:Solubility
chemical_list: D015415:Biomarkers; D006454:Hemoglobins; D008055:Lipids; D023201:CD40 Ligand; D019343:Citric Acid; D006493:Heparin; D004492:Edetic Acid; D001663:Bilirubin