Background: Vitamin B(12) and folate measurements in serum show wide inter-methodology variability. This variability appears to be due in part to the lack of standardisation against internationally accepted reference materials. Pooled human serum, lyophilised in ampoules and designated 03/178, was therefore evaluated by 24 laboratories in seven countries for its suitability to serve as an International Standard (IS) for B(12) and folate.
Methods: IS 03/178 was assayed using a range of commercial analysers, microbiological assays and, for folate, candidate reference methods based on liquid chromatography coupled to isotope-dilution tandem mass spectrometry (LC/MS/MS).
Results: Mean vitamin B(12) and folate values for reconstituted 03/178 across all laboratories and methods were 480 pg/mL [coefficient of variation (CV) 12.8%] and 5.52 ng/mL (CV 17.1%), respectively. The total folate content of reconstituted 03/178, determined using LC/MS/MS, was 12.1 nmol/L (equivalent to 5.33 ng/mL), made up of 9.75 nmol/L 5-methyl tetrahydrofolic acid (5MeTHF; CV 5.5%), 1.59 nmol/L 5-formyl tetrahydrofolic acid (5FoTHF; CV 4.2%) and 0.74 nmol/L folic acid (FA; CV 31.6%). The inclusion of three serum samples in the study with different B(12) and folate levels demonstrated a considerable reduction in inter-laboratory variability when the B(12) and folate content of the samples was determined relative to the IS 03/178 rather than to the analyser calibration. IS 03/178 demonstrated satisfactory long-term stability in accelerated degradation studies.
Conclusions: Use of IS 03/178 to standardise serum B(12) and folate assays reduced inter-laboratory variability. The World Health Organization (WHO) Expert Committee on Biological Standardisation established 03/178 as the first IS for serum vitamin B(12) and serum folate, with assigned values of 480 pg/mL of vitamin B(12) and 12.1 nmol/L folate when the lyophilised contents of the ampoule are reconstituted with 1 mL of water.