*6.1. Development of the T50 Assay*

Circulating biomarkers associated with progression of vascular calcification and mortality in CKD patients lack predictive value. For example, serum levels of fetuin-A and osteoprotegerin positively correlate with mortality of dialysis patients, and soluble klotho is associated with aortic calcification progression [112,113]. In 2012, Pasch et al. introduced a novel concept for the risk assessment for CKD patients. The T50 assay is a measure of the propensity for calcification in blood serum [114], based on the time-dependent shape change of calcium-phosphate precipitation particles. Colloidal spherical-shaped primary calciprotein particles (CPP) convert to crystalline secondary CPPs with radial growth of crystalline needles [115]. Nephelometry allows the determination of the transition step from primary to secondary CPPs. The amount of precipitation depends on the capacity of serum to inhibit this process by calcification inhibitors like fetuin-A. In this assay, the patient's serum is supersaturated by adding 6 mM phosphate and 10 mM calcium to accelerate precipitation. This allows the analysis of the half-maximal transition time (T50). Higher T50 values reflect longer transition times, thereby less propensity for calcification. A potential clinical use needs to be evaluated [114].
