Analytical, experimental and clinical aspects of total 25-hydroxyvitamin D measurement by two ligand assays are differentially biased by disease-related factors

[Display omitted] •Spiking albumin in vitro, differentially biased total-25OHD in CLIA and PBA assays.•Sera of hospitalized patients and outpatients with CKD were studied.•In all clinical groups total-25OHD correlated with albumin and DBP in both assays.•Huge biases were in both patient’s groups, but not in controls.•The PBA might be superior over CLIA in hemodialysis, the best fit is to LC-MS/MS. Pathological concentrations of plasma proteins may confound the results of binding assays. We compared two routinely used total 25-hydroxyvitamin D [t-25(OH)D] methods: a chemiluminescence-immuno-(CLIA) and an electro-chemiluminescence-protein-binding-(ECLPBA) assay. Two sub-studies were performed: 1) In an “in vitro” study, exogenous albumin was added to pools of patients’ sera with low albumin levels; and 2) In “ex vivo” studies of Cohort_1: sera of hospitalized patients with low albumin levels, and of healthy controls; and of Cohort_2: outpatients with chronic kidney disease in pre-dialysis stage, or on peritoneal dialysis and hemodialysis were investigated by the routine and LC_MS/MS methods. When increasing albumin concentrations were “in vitro” added, t-25(OH)D levels were overestimated by ECLPBA, and underestimated by CLIA. In patients’ sera, positive correlations were detected between t-25(OH)D-vitamin D binding protein (DBP) values by both routine methods, and between t-25(OH)D-albumin values by all three methods. Much higher t-25(OH)D was measured by LC_MS/MS in all subgroups. When altering albumin levels with constant DBP concentration, the “in vitro” experiment revealed a higher sensitivity of ECLPBA. The “ex vivo” measurements demonstrated clinically relevant differences between the routine methods. Both routine methods are dependent of the matrix effect in hospitalized patients, which is predicted by the DBP/Albumin ratios. In hemodialysis, ECLPBA is recommended because its outcomes differ less from those of LC_MS/MS. The results of LC_MS/MS are reliable, but not routinely available. A guidance would be valuable on how levels measured by the binding methods differ from those by LC-MS/MS in various clinical conditions.