Blood samples were collected almost daily for routine hematological and biochemical parameters and were analyzed at the central diagnostic laboratory the MUMC, which has acquired ISO 15189 accreditation for these routine measurements.
Study-related blood samples were collected at the following: baseline, day 8 of the study, repopulation, and after the completion of the intervention (day 42). Five ml blood from a serum tube was used for the determination of the serum vitamin C level using a commercial kit from Chromsystems (Gräfelfing, Germany). The intracellular vitamin C concentration of peripheral blood mononuclear cells (PBMCs) was measured by HPLC with UV detection from 8 mL heparinized blood, as described previously (17). This was performed at baseline, repopulation, and at the end of the intervention period using a 4 mL EDTA blood sample for the absolute cell counts of lymphocyte subsets by flowcytometry with BD Trucount tubes (BD Biosciences, Franklin Lakes, NJ, USA) using BD Multitest immunofluorescence reagents (CD3 FITC/CD8 PE/CD45 PerCP/CD4 APC and CD3 FITC/CD16 + CD56 PE/CD45 PerCP/CD19 APC, BD Biosciences) measured on a BD FACSCanto or FACSLyric flow cytometry system (BD Biosciences).
Study-related blood samples were collected at the following: baseline, day 8 of the study, repopulation, and after the completion of the intervention (day 42). Five ml blood from a serum tube was used for the determination of the serum vitamin C level using a commercial kit from Chromsystems (Gräfelfing, Germany). The intracellular vitamin C concentration of peripheral blood mononuclear cells (PBMCs) was measured by HPLC with UV detection from 8 mL heparinized blood, as described previously (17). This was performed at baseline, repopulation, and at the end of the intervention period using a 4 mL EDTA blood sample for the absolute cell counts of lymphocyte subsets by flowcytometry with BD Trucount tubes (BD Biosciences, Franklin Lakes, NJ, USA) using BD Multitest immunofluorescence reagents (CD3 FITC/CD8 PE/CD45 PerCP/CD4 APC and CD3 FITC/CD16 + CD56 PE/CD45 PerCP/CD19 APC, BD Biosciences) measured on a BD FACSCanto or FACSLyric flow cytometry system (BD Biosciences).