- Vitamin D test results vary by 3 to 13 ng depending blood sample tube, stopper – July 2024
- Videos of some of the blood collection tubes, etc,
- VitaminDWiki – Tests for Vitamin D contains:
- VitaminDWiki – Huge variation in Vitamin D test results between 4 testers for 8 people – 2013
- VitaminDWiki – Vitamin D measurements vary with the same sample of blood – March 2014 has a chart
- VitaminDWiki – Big differences (~10 ng) in vitamin D test results, even when using the same type of tester – Aug 2015
- Note: Results vary by: tester, chemicals used, employees, tubes, time of day, blood from arm vs finger, time since last supplement/sun, if D2 was in the blood, epimers in infant blood, etc.
Vitamin D test results vary by 3 to 13 ng depending blood sample tube, stopper – July 2024
Effect of Blood Collection Tubes on Vitamin D Immunoassay Results
Ann Lab Med letter to the editor https://doi.org/10.3343/alm.2024.0234
Hyojin Chae©, M.D., Ph.D.1, Sangyoon Lee©, M.T1, Ae-Ran Choi , M.T.1, Sung-Eun Cho , M.D., Ph.D.2, and Eun-Jee Oh ), M.D., Ph.D.1
1Department of Laboratory Medicine, Seoul St. Mary’s Hospital, College of Medicine, The Catholic University of Korea, Seoul, Korea; 2Department of Endocrine Substance Analysis Center (ESAC), Green Cross Laboratories (GC Labs), Yongin, Korea
Up to 13 ng different test results: either tubes or testers
Dear Editor,
Blood collection tubes are generally considered inert specimen carriers but can potentially induce exogenous interference. Blood collection tubes consist of rubber stoppers, tube-wall materials, separator gels, clot activators or anticoagulants, and surfactants, all of which can interfere with analytical assays [1]. We observed apparent inconsistency in Atellica IM Vitamin D Total assay results (Siemens Healthineers, Forchheim, Germany) across different tubes; therefore, we examined the effect of blood collection tubes on 25-hydroxyvitamin D (25(OH)D) as part of an end-user verification, with approval from the institutional review board (IRB) of Seoul St. Mary's Hospital (IRB No. KC23DSSF0058).
The following four tubes were examined for potential assay interference:
- Greiner Bio-One Vacuette 8-mL serum-separator tube (SST) (Greiner Vacuette; Cat. #455071KR, lot #A2309376 *Greiner Bio-One, Kremsmunster, Austria), Becton Dickinson (BD)
- Vacutainer 8.5-mL SST (BD Vacutainer; Cat. #367528, lot #2237485; BD, Franklin Lakes, NJ, USA),
- AB Medical V-Tube 8-mL tube with clot activator and gel (V-tube; Cat. #301802, lot #8337002; AB Medical, Seoul, Korea), and
- AB Medical VQ-Tube 8-mL with clot activator with thrombin and gel (VQ-tube; Cat. #B0180B, lot #8B41002; AB Medical).
Serum samples from four apparently healthy volunteers (two men and two women; age range, 26-49 yrs) were collected in each of the abovemen- tioned tubes and analyzed for 25(OH)D using the Atellica IM Vitamin D Total assay. Samples collected in Greiner Vacuette tubes exhibited higher values (mean bias, 9.95 ng/mL; range, 4.56-12.34 ng/mL) than those collected in other tubes (Table 1, Fig. 1). This increase would lead to incorrect classification of all four individuals as not deficient in vitamin D, despite having a 25(OH)D level <20 ng/mL (the recommended cutoff value for vitamin D deficiency) [2]. When the samples were analyzed by liquid chromatography-tandem mass spectrometry (LC-MS/MS) with PerkinElmer (Wallac Oy, Turku, Finland) kits, no tube-induced bias (< 5%) was noted. This LC-MS/MS method was certified by the Centers for Disease Control and Prevention Vitamin D Standardization and Certification Program, as previously documented [3].
The tube-induced bias was further examined using the Roche Elecsys Vitamin D Total III (Roche Diagnostics International Ltd,. Rotkreuz, Switzerland) and Atellica IM Vitamin D Total assays. Additionally, serum samples of three other apparently healthy individuals were collected in both Greiner Vacuette and VQ tubes for analysis. Samples collected in Greiner Vacuette tubes exhibited higher 25(OH)D values (mean bias, 6.22 ng/mL) than those collected in VQ tubes when analyzed with the Siemens Atellica IM system. Conversely, no significant bias was observed between the two tubes with the Roche Elecsys Vitamin D Total III assay.
To our knowledge, this is the first report of increased 25(OH)D levels resulting from blood collection in Greiner Vacuette tubes in conjunction with the Atellica IM Vitamin D Total assay. Although the reasons for this observation are not fully understood, tube surfactants, which tend to vary among manufacturers, may affect the vitamin D assay. Notably, tube surfactants are employed to minimize adsorption to the tube wall; however, at sufficiently high concentrations, they may lead to the desorption of antibodies from the solid phase, among other nonspecific effects [4]. The Atellica IM Vitamin D Total assay is a competitive immunoassay that uses anti-fluorescein-labeled mouse monoclonal antibodies covalently bound to paramagnetic particles, a fluorescein-labeled vitamin D analog, and an acridinium esterlabeled anti-25(OH)D mouse monoclonal antibody. Hence, the potential of the surfactant to induce the desorption of antibodies from the solid phase may have lowered the chemiluminescent signal and falsely increased 25(OH)D levels. Notably, a similar bias has been reported for Siemens ADVIA Centaur XP for 25(OH)D when using Vacuette tubes containing a clot activator with or without gel compared to Vacuette tubes with no additives [5, 6]. Because the reagent formulations used in the Atellica IM Analyzer and the ADVIA Centaur system are similar, their interference mechanism may be the same.
Our study is limited by its small sample size. Nevertheless, examining various tubes with specimens obtained from healthy individuals is not readily accessible in routine practice, and our findings clearly show the impact of additives in blood collection tubes on vitamin D immunoassay results. Detecting and preventing tube additive-induced interference poses a unique challenge for clinical laboratories. Timely recognition is difficult because routine QC typically does not include the preanalytical phase, including sample collection and processing in blood collection tubes, in which contact with the tube additives occurs [7]. Therefore, in addition to performing a thorough comparative study, clinical laboratories should be vigilant of any possible changes in assay performance when using new lots and/or different tubes. Furthermore, manufacturers must validate the safety and efficacy of their blood collection tubes through analytical and clinical evaluations, preferably under conditions of maximal interference across all analytical platforms/assays.
Download the PDF from VitaminDWiki
7 REFERENCES
- Bowen RAR, Hortin GL, Csako G, Otaez OH, Remaley AT. Impact of blood collection devices on clinical chemistry assays. Clin Biochem 2010;43: 4-25.
- Holick MF, Binkley NC, Bischoff-Ferrari HA, Gordon CM, Hanley DA, Heaney RP, et al. Evaluation, treatment, and prevention of vitamin D deficiency: an Endocrine Society clinical practice guideline. J Clin Endocrinol Metab 2011;96:1911-30.
- Seo JD, Lee JH, Cho SE, Song J, Lee YW, Yun YM. Evaluation of test standardization of 25-OH vitamin D immunoassays. Lab Med Online 2024; 14:8-16.
- Bowen RAR, Chan Y, Ruddel ME, Hortin GL, Csako G, Demosky SJ Jr., et al. Immunoassay interference by a commonly used blood collection tube additive, the organosilicone surfactant silwet L-720. Clin Chem 2005;51:1874-82.
- Yu S, Cheng X, Fang H, Zhang R, Han J, Qin X, et al. 25OHD analogues and vacuum blood collection tubes dramatically affect the accuracy of automated immunoassays. Sci Rep 2015;5:14636.
- Yu S, Zhou W, Cheng X, Fang H, Zhang R, Cheng Q, et al. Blood collection tubes and storage temperature should be evaluated when using the Siemens ADVIA Centaur XP for measuring 25-hydroxyvitamin D. PLoS One 2016;11:e0166327.
- Kricka LJ, Park JY, Senior MB, Fontanilla R. Processing controls in blood collection tubes reveals interference. Clin Chem 2005;51:2422-3.
Videos of some of the blood collection tubes, etc,
VitaminDWiki – Tests for Vitamin D contains:
- Fact: Many countries no longer pay for more than 1 (some not pay for even a single Vit D test)
They feel that Vit D testing is not needed except for a few conditions (Rickets, etc) Japan is an exception - Fact: Vit D tests are not very accurate
The best lab tests have accuracies and repeatabilities of +-5 ng
Low cost vitamin D Blood Tests - both in lab and at home
Many lab tests have accuracies and repeatabilities of +- 10 ng - or worse
Vitamin D deficiency of a group - 15% to 48% - Fact: Low-cost office/home Vit D tests are available around the world (not US as of 2018)
Low-cost 35 ng Y/N test by Nanospeed
Low-cost Vitamin D testers (two yes-no tests for 11 dollars) - 2024 Nanospeed
Quick, free, self test for deficiency - Fact: 3 major Vit D gene problems are not noticed by Vit D tests
~ 20% of people have poor Vit D genes
Hint that Vit D not getting to cells: Vit D related diseases run in your family
Another hint - you have one of the 40 diseases which are 2X more likely if have poor genes - Fact: A Vit D test will rarely (<1 in 1000) indicate that you are getting too much
- Opinion: If only getting a single test, wait till after supplementing with Vit D
3 months after starting a maintenance dose or 4 weeks after a loading dose
VitaminDWiki – Huge variation in Vitamin D test results between 4 testers for 8 people – 2013
Sometimes there can be a 40 nanogram difference between testers for the same blood sample
Worst case differences for 8 Finns
VitaminDWiki – Vitamin D measurements vary with the same sample of blood – March 2014 has a chart
VitaminDWiki – Big differences (~10 ng) in vitamin D test results, even when using the same type of tester – Aug 2015
Huge differences in % deficient (< 30ng) depending on the tester used
Note: Results vary by: tester, chemicals used, employees, tubes, time of day, blood from arm vs finger, time since last supplement/sun, if D2 was in the blood, epimers in infant blood, etc.
- Endocrine Society recommends for the healthy: no Vitamin D tests, just RDA (many object) - July 2024
- Huge differences between Vitamin D test results: CLIA, LC-MS, and home – June 2024
- How to compare Vitamin D measurements from multiple studies – July 2021
- 4 popular vitamin D testers gave very different results (no surprise) – June 2021
- Epimers of Vitamin D: A Review – Jan 2020
- Vitamin D testing accuracies, including dried blood spot – Jan 2020
- Vitamin D testers have different test results if there is chronic kidney disease – Sept 2019
- Vitamin D levels observed to vary by 20 percent during a day – Feb 2019
- Problems with Vitamin D Testing – chapter – Aug 2019
- Vitamin D deficiency of a group - 15 pcnt to 48 pcnt (depends on tester used) - Nov 2017