Accessible Unlicensed Requires Authentication Published by De Gruyter March 1, 2007

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. Thorpe, Alan Heath, Sheena Blackmore, Anne Lee, Malcolm Hamilton, Sean O'Broin, Bryant C. Nelson and Christine Pfeiffer
From the journal


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.


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,


1. Working Party of the BCSH General Haematology Task Force. Guidelines on the investigation and diagnosis of cobalamin and folate deficiencies. Clin Lab Haematol 1994;16:101–15.Search in Google Scholar

2. Solomon LR. Cobalamin responsive disorders in the ambulatory care setting: unreliability of cobalamin, methylmalonic acid and homocysteine testing. Blood2005;105:978–85.Search in Google Scholar

3. Goringe A, Ellis R, McDowell I, Vidal-Alaball J, Jenkins C, Butler C, et al. The limited value of methylmalonic acid, homocysteine, and holotranscobalamin in the diagnosis of early B12 deficiency. Haematologica2006;91:231–4.Search in Google Scholar

4. Curtis AD, Mussett MV, Kennedy DA. British Standard for human serum vitamin B12. Clin Lab Haematol1986;8:135–47.Search in Google Scholar

5. Gunter EW, Bowman BA, Caudill SP, Twite DB, Adams MJ, Sampson EJ. Results of an international round robin for serum and whole-blood folate. Clin Chem1996;42:1689–94.Search in Google Scholar

6. Thorpe SJ, Sands D, Heath AB, Hamilton MS, Blackmore S, Barrowcliffe T. An International Standard for whole blood folate: evaluation of a lyophilised haemolysate in an international collaborative study. Clin Chem Lab Med2004;42:533–9.Search in Google Scholar

7. Satterfield MB, Sniegoski LT, Sharpless KF, Welch MJ, Hornikova A, Zhang NF, et al. Development of a new standard reference material: SRM 1955 (homocysteine and folate in human serum). Anal Bioanal Chem2006;385:612–22.Search in Google Scholar

8. Nelson BC, Pfeiffer CM, Margolis SA, Nelson CP. Solid-phase extraction-electrospray ionization mass spectrometry for the quantification of folate in human plasma or serum. Anal Biochem2004;325:41–51.Search in Google Scholar

9. Pfeiffer CM, Fazili Z, McCoy L, Zhang M, Gunter EW. Determination of folate vitamers in human serum by stable-isotope-dilution tandem mass spectrometry and comparison with radioassay and microbiologic assay. Clin Chem2004;50:423–32.Search in Google Scholar

10. Kelleher BP, O'Broin SD. Microbiological assay for vitamin B12 performed in 96-well microtitre plates. J Clin Pathol1991;44:592–5.Search in Google Scholar

11. O'Broin S, Kelleher B. Microbiological assay on microtitre plates of folate in serum and red cells. J Clin Pathol1992;45:344–7.Search in Google Scholar

12. Kirkwood TB. Predicting the stability of biological standards and products. Biometrics1977;33:736–42.Search in Google Scholar

13. WHO. Recommendations for the preparation, characterization and establishment of international and other biological reference materials. WHO/BS/04.1995 (revised 2004). Geneva: World Health Organization.Search in Google Scholar

14. In vitro diagnostic devices – measurement of quantities in biological samples – metrological traceability of values assigned to calibrators and control materials. EN ISO 17511. ISO, 2003.Search in Google Scholar

Published Online: 2007-03-01
Published in Print: 2007-03-01

©2007 by Walter de Gruyter Berlin New York