Varo Nerea, Nuzzo Rebecca, Natal Cristina, Libby Peter, Schönbeck Uwe
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.
Clin Sci (Lond). 2006 Nov;111(5):341-7. doi: 10.1042/CS20060047.
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.
促动脉粥样硬化介质CD40L（CD40配体）的可溶性形式已成为心血管事件的诊断和预后标志物。然而，由于血小板在激活时会释放CD40L，准确测量已被证明具有挑战性。本研究解决了关于血液可溶性CD40L（sCD40L）实验室评估的合适标本和制备方法的争议。从健康志愿者（n = 20）中采集血清和血浆（用乙二胺四乙酸、柠檬酸盐或肝素采集），并立即或在一至三个冻融循环后以及在不同离心速度下通过酶联免疫吸附测定法分析sCD40L。在血浆sCD40L水平低和高的受试者中测量尿sCD40L水平。血清sCD40L水平（5.45±4.55 ng/ml；P<0.001）高于柠檬酸盐、乙二胺四乙酸或肝素血浆中的水平（分别为1.03±1.07、1.43±1.03或1.80±1.25 ng/ml），血浆制剂之间无显著差异。增加重力值（200 - 13000 g）会逐渐耗尽血浆中的血小板，导致sCD40L水平降低。重复冻融循环显著（P<0.05）增加富含血小板但非血小板耗尽血浆中的sCD40L浓度（高达2.4倍）。胆红素和血红蛋白产生正向干扰，三酰甘油（甘油三酯）和胆固醇会淬灭CD40L信号。尿样中未检测到sCD40L。总之，血清产生的sCD40L浓度高于血浆；sCD40L的准确测量需要排除血小板并避免其事后激活。应排除胆红素、血红蛋白和/或三酰甘油浓度高的样本，因为这些物质会干扰检测。
