Comparison of three different methods for 25(oh)-vitamin d determination and vitamin d status in general population – Serbian experience / Poređenje tri različite metode za određivanje 25(oh)-vitamina D i statusa vitamina D u opštoj populaciji – srpsko iskustvo

Open access


Determination of 25-hydroxyvitamin D [25(OH)D] represents a unique challenge, considering its lipophilic na - ture. Considering the widespread prevalence of vitamin D deficiency, which leads to increasing number of requests for 25(OH)D determination, immunoassay measurements adjusted to automated analyzers are being developed. Because of the variability among assays, it is often difficult to monitor vitamin D status and supplementation. The aim of this study was to compare the results of two immunoassays with high performance liquid chromatography with ultraviolet detection (HPLC-UV). Also, the aim was to estimate vitamin D status, since up to date the prevalence of vitamin D deficiency in Serbia was not examined. We have evaluated analytical characteristics of two automated immunoassays for 25(OH)D determination, from Roche (CobasR e601) and Abbott (Architect). For comparison studies we used HPLC analysis of 25-(OH)-Vitamin D3/D2 from Chromsystems (Waters isocratic system). In order to estimate vitamin D status in general population, we have searched the database of the laboratory information system and analyzed the data from 533 patients whose 25(OH)D was determined together with intact parathyroid hormone (iPTH). For imprecision assessment, four serum pools were prepared with following 25(OH)D concentrations: 35 nmol/L, ∼50 nmol/L, ∼75 nmol/L and ∼125 nmol/L. Obtai ned CVs for Roche method were 1.5-2.8% for within-run and 4.0-6.7% for between-run imprecision. For Abbott method, CVs were 0.7-4.4% for within- run and 3.8-7.2% for between-run imprecision. Inaccuracy was analyzed with commercial control sera. Obtained deviations from target value were 2.1% for Roche assay and 1.3-1.5% for Abbott method, and were not statistically significant (P>0.05). Comparison of Roche and HPLC-UV methods using Passing-Bablok regression analysis gave the following equation for the regression line y=0.937x+9.518 (r=0.739; n=97) and the regression line equation from the comparison of Abbott and HPLC-UV methods was y=0.745x+10.343 (r=0.793; n=97). Mean difference and SD for Bland-Altman plot were -4.5 nmol/L and 21.75 nmo/L, respectively for Roche method and 6.4 nmol/L and 18.8 nmol/L, respectively for Abbott. Statistical analysis (Chi-square test) of frequency distribution among different vitamin D status categories (<25 nmol/L severe deficiency, 25-50 nmol/L deficiency, 50-75 nmol/L insufficiency and >75 nmol/L sufficiency) showed that the frequency distribution obtained with Abbott method was significantly different from the distribution of the HPLC results, in contrast to Roche results frequency distribution which did not differ significantly. Also, statistical analysis of the agreement between the three methods for each vitamin D status category showed that results of both Roche and Abbott methods were significantly higher than HPLC in the two deficiency categories (P=0.005 for Roche, P=0.0407 for Abbott), and in the sufficiency category Abbott method significantly underestimated concentration of 25(OH)D compared to HPLC results (P<0.0001). Median population values of 25(OH)D and iPTH were 41.8 nmol/L and 76.6 ng/L, respectively. ANOVA analyses showed significant (P<0.05) decrease in iPTH and Ca2+ concentrations across the 25(OH)D concentration categories. Stepwise multiple linear regression analysis indicated independent correlation of iPTH with 25(OH)D concentration (b=-0.290, P=0.0008). Also, one-way ANOVA with Student-Newman-Keuls test demonstrated that 25(OH)D concentrations measured in summer and autumn were significantly (P<0.001) higher compared to those determined in winter and spring. Despite acceptable imprecision and inaccuracy of both examined methods, results obtained with them did not correlate well with HPLC-UV (r<0.9), which was used as a reference. However, methods showed satisfactory ability to classify patients into vitamin D status categories, which is important for diagnosis of vitamin D deficiency and therapy follow-up. About two thirds (68.5%) of the examined po pulation had vitamin D deficiency (25(OH)D<50 nmol/L) and only 8% had sufficient 25(OH)D concentration (>75 nmol/L).

If the inline PDF is not rendering correctly, you can download the PDF file here.

  • 1. Holick MF. Vitamin D deficiency. N Engl J Med 2007; 357: 266-81.

  • 2. Kleerekoper M Schleicher RL Eisman J Bouillon R Singh RJ Holick MF. Clinical applications for vitamin D assays: what is known and what is wished for. Clin Chem 2011; 57(9): 1227-32.

  • 3. Endres DB Rude RK. Mineral and bone metabolism. In: Burtis CA Ashwood ER Bruns DE editors. Tietz Textbook of Clinical Chemistry. 4th ed. Philadelphia: WB Saunders 2006: 1929-6.

  • 4. Wallace AH Gibson S de la Hunty A Lamberg-Allardt C Ashwell M. Measurement of 25-hydroxyvitamin D in the clinical laboratory: current procedures performance characteristics and limitations. Steroids 2010; 75: 477-88.

  • 5. Tai SS Bedner M Phinney KW. Development of a candidate reference measurement procedure for the determination of 25-hydroxyvitamin D3 and 25-hydroxyvitamin D2 in human serum using isotope-dilution liquid chromatography- tandem mass spectrometry. Anal Chem 2010; 82: 1942-8.

  • 6. Stepman HC Vanderroost A Van Uytfanghe K Thien - pont LM. Candidate reference measurement procedures for serum 25-hydroxyvitamin D3 and 25-hydroxyvitamin D2 by using isotope-dilution liquid chromatography-tandem mass spectrometry. Clin Chem 2011; 57: 441-8.

  • 7. Beastall G Rainbow S. Vitamin D reinvented: implications for clinical chemistry. Clin Chem 2008; 54(4): 630-2.

  • 8. Carter GD. 25-hydroxyvitamin D assays: the quest for accuracy. Clin Chem 2009; 55(7): 1300-2.

  • 9. Binkley N Krueger D Cowgill CS Plum L Lake E Hansen KE et al. Assay variation confounds the diagnosis of hypovitaminosis D: call for standardization. J Clin Endocrinol Metab 2004; 89: 3152-7.

  • 10. Holick MF. The D-lemma: to screen or not to screen for 25-hydroxyvitamin D concentrations. Clin Chem 2010; 56(5): 729-31.

  • 11. Passing H Bablok W. A new biometrical procedure for testing the equality of measurements from two different analytical methods. Application of linear regression procedures procedures for method comparison studies in clinical chemistry Part I. J Clin Chem Clin Biochem 1983; 21: 709-20.

  • 12. Bland JM Altman DG. Statistical methods for assessing agreement between two methods of clinical measurement. Lancet 1986; i: 307-10.

  • 13. Stockl D Sluss PM Thienpont LM. Specifications for trueness and precision of a reference measurement system for serum/plasma 25-hydroxyvitamin D analysis. Clin Chem Acta 2009; 408: 8-13.

  • 14. Bossuyt PMM. Defining biomarker performance and clinical validity. Journal of Medical Biochemistry 2011; 30: 193-200.

  • 15. Thienpont LM Stepman HCM Vesper HW. Standar - dization of measurements of 25-Hydroxyvitamin D3 and D2. Scand J Clin Lab Invest 2012; 72: Suppl 243: 41-9.

  • 16. Sempos CT Vesper HW Phinney KW Thienpont LM Coates PM. Vitamin D status as an international issue: National surveys and the problem of standardization. Scand J Clin Lab Invest 2012; 72: Suppl 243: 32-40.

  • 17. Gonzalez-Molero I Morcillo S Valdes S et al. Vitamin D deficiency in Spain: population-based cohort study. European Journal for Clinical Nutrition 2011; 65: 321-8.

  • 18. Hypponen E Power C. Hypovitaminosis D in British adults at age 45 y: nationwide cohort study of dietary and lifestyle predictors. Am J Clin Nutr 2007; 85: 860-8.

  • 19. Christensen MHE Lien EA Hustad S et al. Seasonal and age-related differences in serum 25-hydroxyvitamin D 125-dihydroxyvitamin D and parathyroid hormone in patients from Western Norway. Scandinavian Journal of Clinical and Laboratory Investigation 2010; 70: 281-6.

  • 20. Macdonald HM Mavroeidi A Barr RJ et al. Vitamin D status in postmenopausal women living at higher latitudes in the UK in relation to bone health overwight sunlight exposure and dietary vitamin D. Bone 2008; 42: 996-1003.

  • 21. Holick MF. High prevalence of vitamin D inadequacy and implications for health. Mayo Clin Proc 2006; 81(3): 353-73.

  • 22. Rollins G. The new vitamin D. Clinical Laboratory News 2007; 33 (12).

  • 23. MacLaughlin J Holick MF. Aging decrease the capacity of human skin to produce vitamin D3. J Clin Invest 1985; 76: 1536-8.

  • 24. Holick MF Matsuoka LY Wortsman J. Age vitamin D and solar ultraviolet. Lancet 1989; 2: 1104-5.

Journal information
Impact Factor

IMPACT FACTOR 2018: 2.000
5-year IMPACT FACTOR: 1.075

CiteScore 2018: 1.47

SCImago Journal Rank (SJR) 2018: 0.523
Source Normalized Impact per Paper (SNIP) 2018: 0.581

Cited By
All Time Past Year Past 30 Days
Abstract Views 0 0 0
Full Text Views 228 66 3
PDF Downloads 95 42 4