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Clinical Chemistry and Laboratory Medicine (CCLM)

Published in Association with the European Federation of Clinical Chemistry and Laboratory Medicine (EFLM)

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International Standard for serum vitamin B12 and serum folate: international collaborative study to evaluate a batch of lyophilised serum for B12 and folate content

Susan J. Thorpe1 / Alan Heath2 / Sheena Blackmore3 / Anne Lee4 / Malcolm Hamilton5 / Sean O'Broin6 / Bryant C. Nelson7 / Christine Pfeiffer8









Corresponding author: Dr. S.J. Thorpe, Parenterals Section, Biotherapeutics Group, National Institute for Biological Standards and Control, Blanche Lane, South Mimms, Potters Bar, Hertfordshire EN6 3QG, UK Phone: +44-1707-641251, Fax: +44-1707-641053,

Citation Information: Clinical Chemical Laboratory Medicine. Volume 45, Issue 3, Pages 380–386, ISSN (Online) 1437-4331, ISSN (Print) 1434-6621, DOI: 10.1515/CCLM.2007.072, March 2007

Publication History

October 23, 2006
December 20, 2006


Background: Vitamin B12 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 B12 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 B12 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 B12 and folate levels demonstrated a considerable reduction in inter-laboratory variability when the B12 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 B12 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 B12 and serum folate, with assigned values of 480 pg/mL of vitamin B12 and 12.1 nmol/L folate when the lyophilised contents of the ampoule are reconstituted with 1 mL of water.

Clin Chem Lab Med 2007;45:380–6.

Keywords: folate; liquid chromatography coupled to isotope-dilution tandem mass spectrometry (LC/MS/MS); 5-methyl tetrahydrofolic acid (5MeTHF); reference; standardisation; vitamin B12

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