Skip to main content
U.S. flag

An official website of the United States government

Official websites use .gov
A .gov website belongs to an official government organization in the United States.

Secure .gov websites use HTTPS
A lock ( ) or https:// means you’ve safely connected to the .gov website. Share sensitive information only on official, secure websites.

Assessment of Serum Total 25-Hydroxyvitamin D Assays for Vitamin D External Quality Assessment Scheme (DEQAS) Materials Distributed at Ambient and Frozen Conditions



Johanna Camara, Christopher T. Sempos, Emma L. Williams, Graham D. Carter, Julia C. Jones, Carolyn Burdette, Grace Hahm, Federica Nalin, David L. Duewer, Adam J. Kuszak, J. M. Betz, Andrew Hoofnagle, Pierre Lukas, E. Cavalier, Ramon Durazo-Arvizu, P. M. Crump, Christian Popp, Christian Beckert, Glen Van Slooten, Patrick Gradon, Joelle Morgand, Sarah Paul, Bruno Emanuelli, Angelo Maggio, Fiona Ivison, Ralf Fischer, Jody M. van den Ouweland, Chung S. Ho, Emmett W. Law, Jean-Nicolas Simard, Renaud Gonthier, Brett Holmquist, Manisha S. Patwardhan, Marcelo C. Batista, Heather Pham, Sarah Meadows, Eugene Jansen, Dilshad A. Khan, Kimberly Robyak, Mark Kilbane, Neil Parker, Vincent Chen, Robert Fitzgerald, Sohail Mushtaq, Michael W. Clarke, Norma Breen, Christine Simpson, Stephen A. Wise


The Vitamin D External Quality Assessment Scheme (DEQAS) distributes human serum samples four times per year to over 1000 participants worldwide for the determination of total serum 25-hydroxyvitamin D [25(OH)D)]. These samples are stored at −40 °C prior to distribution and the participants are instructed to store the samples frozen at −20 °C or lower after receipt; however, the samples are shipped to participants at ambient conditions (i.e., no temperature control). To address the question of whether shipment at ambient conditions is sufficient for reliable performance of various 25(OH)D assays, the equivalence of DEQAS human serum samples shipped under frozen and ambient conditions was assessed. As part of a Vitamin D Standardization Program (VDSP) commutability study, two sets of the same nine DEQAS samples were shipped to participants at ambient temperature and frozen on dry ice. Twenty-eight laboratories participated in this study and provided 34 sets of results for the measurement of 25(OH)D using 20 ligand binding assays and 14 liquid chromatography–tandem mass spectrometry (LC–MS/MS) methods. Equivalence of the assay response for the frozen versus ambient DEQAS samples for each assay was evaluated using multi-level modeling, paired t-tests including a false discovery rate (FDR) approach, and ordinary least squares linear regression analysis of frozen versus ambient results. Using the paired t-test and confirmed by FDR testing, differences in the results for the ambient and frozen samples were found to be statistically significant at p < 0.05 for four assays (DiaSorin, DIAsource, Siemens, and SNIBE prototype). For all 14 LC–MS/MS assays, the differences in the results for the ambient- and frozen-shipped samples were not found to be significant at p < 0.05 indicating that these analytes were stable during shipment at ambient conditions. Even though assay results have been shown to vary considerably among different 25(OH)D assays in other studies, the results of this study also indicate that sample handling/transport conditions may influence 25(OH)D assay response for several assays.
Analytical and Bioanalytical Chemistry


25-Hydroxyvitamin D3, 25-Hydroxyvitamin D2, Total 25-hydroxyvitamin D, Liquid chromatography–tandem mass spectrometry (LC–MS/MS), Vitamin D External Quality Assessment Scheme (DEQAS), Ligand binding assay


Camara, J. , Sempos, C. , Williams, E. , Carter, G. , Jones, J. , Burdette, C. , Hahm, G. , Nalin, F. , Duewer, D. , Kuszak, A. , Betz, J. , Hoofnagle, A. , Lukas, P. , Cavalier, E. , Durazo-Arvizu, R. , Crump, P. , Popp, C. , Beckert, C. , Van Slooten, G. , Gradon, P. , Morgand, J. , Paul, S. , Emanuelli, B. , Maggio, A. , Ivison, F. , Fischer, R. , van den Ouweland, J. , Ho, C. , Law, E. , Simard, J. , Gonthier, R. , Holmquist, B. , Patwardhan, M. , Batista, M. , Pham, H. , Meadows, S. , Jansen, E. , Khan, D. , Robyak, K. , Kilbane, M. , Parker, N. , Chen, V. , Fitzgerald, R. , Mushtaq, S. , Clarke, M. , Breen, N. , Simpson, C. and Wise, S. (2021), Assessment of Serum Total 25-Hydroxyvitamin D Assays for Vitamin D External Quality Assessment Scheme (DEQAS) Materials Distributed at Ambient and Frozen Conditions, Analytical and Bioanalytical Chemistry, [online], (Accessed May 24, 2024)


If you have any questions about this publication or are having problems accessing it, please contact

Created November 9, 2021, Updated November 10, 2021