Context: Measurement of circulating 25-hydroxyvitamin D (25(OH)D) is the accepted clinical indicator of vitamin D status. However, between-laboratory differences in measurement of this analyte exist, which may confound clinical care.

Objective: We investigated current agreement of 25(OH)D measurement in clinical laboratories and explored the possibility that simple calibration would improve between-laboratory agreement.

Design and Participants: Serum obtained from healthy volunteers (age 20–60 years) and one "calibrator," selected to have a 25(OH)D value near 30 ng/ml, were sent for 25(OH)D measurement in four clinical laboratories (labs A-D) using HPLC, LCMSMS and RIA methodologies.

Main Outcome Measure: Serum 25(OH)D; based upon self-report, the laboratory with the lowest inter-assay %CV was assigned as the reference to which the others were compared using linear regression and Bland-Altman analyses.

Results: Good correlation was observed for 25(OH)D measurement between laboratory A and laboratories B-D; R² = 0.99, 0.81 and 0.95 respectively. Modest between-laboratory variation was noted; the mean bias ranged from 2.9–5.2 ng/ml. Consistent with a systematic offset, each value in laboratory B was higher than in laboratory A and 89% of values from laboratories B-D were higher than laboratory A. Use of a single calibrator and correction factor reduced mean between-laboratory bias for labs B and D.

Conclusion: Measurement of 25(OH)D by clinical laboratories yields similar results. Use of even a single calibrator will improve, but not resolve, between-laboratory variability. Based upon these data, in combination with reported within-individual variability, we recommend that clinicians aim for values greater than 30 ng/ml in their patients.