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.
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
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