15289-s-bos

74 | Chapter 5 Materials and Methods Study population Between February 2008 and June 2011, 145 patients with a diagnosis of FH were recruited from the outpatient clinic, Erasmus Medical Center, Rotterdam, The Netherlands. As previously described [17], the inclusion criteria for FH were: either patients with a documented LDL receptor mutation or patients with LDL-cholesterol > 95th percentile for gender and age in combination with either 1) the presence of typical tendon xanthomas in the patient or in a 1st degree relative or 2) LDL-cholesterol > 95th percentile for gender and age in a 1st degree relative or 3) proven coronary artery disease in a 1st degree relative under age of 60. Patients with previous symptomatic CVD or with symptoms suggestive of ischemic heart disease at the time of inclusion were excluded from the study. All participants provided written informed consent prior to inclusion (Reference number MEC 2007-183). Of 129 FH patients, plasma samples were available for Lp(a) measurement; these patients were included in this study. Computed tomographic scanning and calcification score measurement All computed tomographic (CT) scannings were performed using a dual-source CT scanner (Somatom Definition, Siemens Medical Solutions, Forchheim, Germany). AVC and coronary artery calcification (CAC) were determined as previously described [17]. The calcium scores were calculated and expressed as Agatston Units (AU) as previously described [18]. An AU score of more than 400 was regarded as an extensive calcification [19]. Lp(a) measurement Venous blood samples were collected after an overnight fast. After centrifugation, plasma samples and buffy coats were collected and stored at -80oC until analysis. Lipid parameters were measured by standard laboratory techniques. Plasma Lp(a) concentrations were measured using a particle enhanced immunoturbidimetric assay, independently of apo(a) KIV repeats (Diagnostic System #171399910930) [20]. In the samples with low Lp(a) concentration, Lp(a) levels were determined using an enzyme- linked immunosorbent assay (ELISA) [21] that has a markedly lower detection limit. Detection of apolipoprotein(a) KIV repeats The number of apo(a) KIV repeats was determined by immunoblotting, using a volume