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.
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November 2006
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Research Article|
October 13 2006
Influence of pre-analytical and analytical factors on soluble CD40L measurements
Nerea Varo;
Nerea Varo
*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, U.S.A.
†Center for Applied Medical Research, Avenida Pío XII 55, Pamplona, Spain
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Rebecca Nuzzo;
Rebecca Nuzzo
*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, U.S.A.
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Cristina Natal;
Cristina Natal
†Center for Applied Medical Research, Avenida Pío XII 55, Pamplona, Spain
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Peter Libby;
*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, U.S.A.
Correspondence: Professor Peter Libby (email [email protected]).
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Uwe Schönbeck
Uwe Schönbeck
‡Boehringer Ingelheim Pharmaceutical, Inc., Ridgefield, CT 06877, U.S.A.
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Publisher: Portland Press Ltd
Received:
February 23 2006
Revision Received:
June 02 2006
Accepted:
July 19 2006
Accepted Manuscript online:
July 19 2006
Online ISSN: 1470-8736
Print ISSN: 0143-5221
The Biochemical Society
2006
Clin Sci (Lond) (2006) 111 (5): 341–347.
Article history
Received:
February 23 2006
Revision Received:
June 02 2006
Accepted:
July 19 2006
Accepted Manuscript online:
July 19 2006
Citation
Nerea Varo, Rebecca Nuzzo, Cristina Natal, Peter Libby, Uwe Schönbeck; Influence of pre-analytical and analytical factors on soluble CD40L measurements. Clin Sci (Lond) 1 November 2006; 111 (5): 341–347. doi: https://doi.org/10.1042/CS20060047
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