Skip to main content Accessibility help

Application of dried blood spots to determine vitamin D status in a large nutritional study with unsupervised sampling: the Food4Me project

  • Ulrich Hoeller (a1), Manuela Baur (a1), Franz F. Roos (a1), Lorraine Brennan (a2), Hannelore Daniel (a3), Rosalind Fallaize (a4), Hannah Forster (a2), Eileen R. Gibney (a2), Mike Gibney (a2), Magdalena Godlewska (a5), Kai Hartwig (a3), Silvia Kolossa (a3), Christina P. Lambrinou (a6), Katherine M. Livingstone (a7), Julie A. Lovegrove (a4), Anna L. Macready (a4), Yannis Manios (a6), Cyril F. M. Marsaux (a8), J. Alfredo Martinez (a9) (a10), Carlos Celis-Morales (a7), George Moschonis (a6), Santiago Navas-Carretero (a9) (a10), Clare B. O’Donovan (a2), Rodrigo San-Cristobal (a9) (a10), Wim H. M. Saris (a8), Agnieszka Surwiłło (a5), Iwona Traczyk (a5), Lydia Tsirigoti (a6), Marianne C. Walsh (a2), Clara Woolhead (a2), John C. Mathers (a7) and Peter Weber (a1)...


An efficient and robust method to measure vitamin D (25-hydroxy vitamin D3 (25(OH)D3) and 25-hydroxy vitamin D2 in dried blood spots (DBS) has been developed and applied in the pan-European multi-centre, internet-based, personalised nutrition intervention study Food4Me. The method includes calibration with blood containing endogenous 25(OH)D3, spotted as DBS and corrected for haematocrit content. The methodology was validated following international standards. The performance characteristics did not reach those of the current gold standard liquid chromatography-MS/MS in plasma for all parameters, but were found to be very suitable for status-level determination under field conditions. DBS sample quality was very high, and 3778 measurements of 25(OH)D3 were obtained from 1465 participants. The study centre and the season within the study centre were very good predictors of 25(OH)D3 levels (P<0·001 for each case). Seasonal effects were modelled by fitting a sine function with a minimum 25(OH)D3 level on 20 January and a maximum on 21 July. The seasonal amplitude varied from centre to centre. The largest difference between winter and summer levels was found in Germany and the smallest in Poland. The model was cross-validated to determine the consistency of the predictions and the performance of the DBS method. The Pearson’s correlation between the measured values and the predicted values was r 0·65, and the sd of their differences was 21·2 nmol/l. This includes the analytical variation and the biological variation within subjects. Overall, DBS obtained by unsupervised sampling of the participants at home was a viable methodology for obtaining vitamin D status information in a large nutritional study.


Corresponding author

* Corresponding author: U. Hoeller, email


Hide All
1. Demirev, PA (2013) Dried blood spots: analysis and applications. Anal Chem 85, 779789.
2. McDade, TW, Williams, S & Snodgrass, JJ (2007) What a drop can do: dried blood spots as a minimally invasive method for integrating biomarkers into population-based research. Demography 44, 899925.
3. Celis-Morales, C, Livingstone, K, Marsaux, CM, et al. (2014) Design and baseline characteristics of the Food4Me study: a web-based randomised controlled trial of personalised nutrition in seven European countries. Genes Nutr 10, 113.
4. Guthrie, R & Susi, A (1963) A simple phenylalanine method for detecting phenylketonuria in large populations of newborn infants. Pediatrics 32, 338343.
5. Tanna, S & Lawson, G (2011) Analytical methods used in conjunction with dried blood spots. Anal Methods 3, 17091718.
6. Shi, H, Ma, Y, Humphrey, JH, et al. (1995) Determination of vitamin A in dried human blood spots by high-performance capillary electrophoresis with laser-excited fluorescence detection. J Chromatogr B Biomed Appl 665, 8996.
7. Craft, NE, Haitema, T, Brindle, LK, et al. (2000) Retinol analysis in dried blood spots by HPLC. J Nutr 130, 882885.
8. O’Broin, SD & Kelleher, BP (2008) A dried serum spot assay for vitamin B12 . Clin Chem Lab Med 46, 354358.
9. Eyles, D, Anderson, C, Ko, P, et al. (2009) A sensitive LC/MS/MS assay of 25OH vitamin D3 and 25OH vitamin D2 in dried blood spots. Clin Chim Acta 403, 145151.
10. Eyles, DW, Morley, R, Anderson, C, et al. (2010) The utility of neonatal dried blood spots for the assessment of neonatal vitamin D status. Paediatr Perinat Epidemiol 24, 303308.
11. Newman, MS, Brandon, TR, Groves, MN, et al. (2009) A liquid chromatography/tandem mass spectrometry method for determination of 25-hydroxy vitamin D2 and 25-hydroxy vitamin D3 in dried blood spots: a potential adjunct to diabetes and cardiometabolic risk screening. J Diabetes Sci Technol 3, 156162.
12. Higashi, T, Suzuki, M, Hanai, J, et al. (2011) A specific LC/ESI-MS/MS method for determination of 25-hydroxyvitamin D3 in neonatal dried blood spots containing a potential interfering metabolite, 3-epi-25-hydroxyvitamin D3 . J Sep Sci 34, 725732.
13. Larkin, EK, Gebretsadik, T, Koestner, N, et al. (2011) Agreement of blood spot card measurements of vitamin D levels with serum, whole blood specimen types and a dietary recall instrument. PLoS ONE 6, e16602.
14. Motohara, K, Endo, F & Matsuda, I (1987) Screening for late neonatal vitamin K deficiency by acarboxyprothrombin in dried blood spots. Arch Dis Child 62, 370375.
15. O’Broin, S & Gunter, E (2002) Dried-serum spot assay for folate. Clin Chem 48, 11281130.
16. Erhardt, JG, Craft, NE, Heinrich, F, et al. (2002) Rapid and simple measurement of retinol in human dried whole blood spots. J Nutr 132, 318321.
17. Bikle, DD, Gee, E, Halloran, B, et al. (1986) Assessment of the free fraction of 25-hydroxyvitamin D in serum and its regulation by albumin and the vitamin D-binding protein. J Clin Endocrinol Metab 63, 954959.
18. Al-oanzi, ZH, Tuck, SP, Raj, N, et al. (2006) Assessment of vitamin D status in male osteoporosis. Clin Chem 52, 248254.
19. Livingstone, KM, Celis-Morales, C, Navas-Carretero, S, et al. (2015) Profile of European adults interested in internet-based personalised nutrition: the Food4Me study. Eur J Nutr (epublication ahead of print version 17 April 2015).
20. Lauridsen, C, Halekoh, U, Larsen, T, et al. (2010) Reproductive performance and bone status markers of gilts and lactating sows supplemented with two different forms of vitamin D. J Anim Sci 88, 202213.
21. European Medicines Agency (2011) Guideline on bioanalytical method validation. EMEA/CHMP/EWP/192217/2009. London: European Medicines Agency.
22. Timmerman, P, White, S, Globig, S, et al. (2011) EBF recommendation on the validation of bioanalytical methods for dried blood spots. Bioanalysis 3, 15671575.
23. Bendik, I, Friedel, A, Roos, FF, et al. (2014) Vitamin D: a critical and essential micronutrient for human health. Front Physiol 5, 248.
24. Bischoff-Ferrari, H, Dawson-Hughes, B, Stocklin, E, et al. (2012) Oral supplementation with 25(OH)D3 versus vitamin D3: effects on 25(OH)D levels, lower extremity function, blood pressure, and markers of innate immunity. J Bone Miner Res 27, 160169.
25. R Core Team (2013) R: A Language and Environment for Statistical Computing. Vienna, Austria: R Foundation for Statistical Computing.
26. Pinheiro, J, Bates, D, DebRoy, S, et al. (2014) nlme: Linear and Nonlinear Mixed Effects Models. R package version 3.1–117.
27. Wahl, DA, Cooper, C, Ebeling, PR, et al. (2012) A global representation of vitamin D status in healthy populations. Arch Osteoporos 7, 155172.
28. Thirup, P (2003) Haematocrit: within-subject and seasonal variation. Sports Med 33, 231243.
29. De Kesel, PM, Sadones, N, Capiau, S, et al. (2013) Hemato-critical issues in quantitative analysis of dried blood spots: challenges and solutions. Bioanalysis 5, 20232041.
30. Capiau, S, Stove, VV, Lambert, WE, et al. (2013) Prediction of the hematocrit of dried blood spots via potassium measurement on a routine clinical chemistry analyzer. Anal Chem 85, 404410.
31. Kvaskoff, D, Ko, P, Simila, HA, et al. (2012) Distribution of 25-hydroxyvitamin D3 in dried blood spots and implications for its quantitation by tandem mass spectrometry. J Chromatogr B Analyt Technol Biomed Life Sci 901, 4752.
32. Celis-Morales, C, Livingstone, KM, Woolhead, C, et al. (2015) How reliable is internet-based self-reported identity, socio-demographic and obesity measures in European adults? Genes Nutr 10, 476.
33. Smith, JE & Goodman, DS (1971) Turnover and transport of vitamin D and of a polar metabolite with the properties of 25-hydroxycholecalciferol in human plasma. J Clin Invest 50, 21592167.
34. Hypponen, E & Power, C (2007) Hypovitaminosis D in British adults at age 45 y: nationwide cohort study of dietary and lifestyle predictors. Am J Clin Nutr 85, 860868.
35. Zittermann, A, Scheld, K & Stehle, P (1998) Seasonal variations in vitamin D status and calcium absorption do not influence bone turnover in young women. Eur J Clin Nutr 52, 501506.
36. Hintzpeter, B, Mensink, GBM, Thierfelder, W, et al. (2008) Vitamin D status and health correlates among German adults. Eur J Clin Nutr 62, 10791089.



Altmetric attention score

Full text views

Total number of HTML views: 0
Total number of PDF views: 0 *
Loading metrics...

Abstract views

Total abstract views: 0 *
Loading metrics...

* Views captured on Cambridge Core between <date>. This data will be updated every 24 hours.

Usage data cannot currently be displayed