Vitamin D test result can vary by 40 ng (10 reasons) – July 2024

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

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Up to 13 ng different test results: either tubes or testers

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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
  1. 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.
  2. 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.
  3. 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.
  4. 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.
  5. 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.
  6. 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.
  7. 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:

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
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VitaminDWiki – Vitamin D measurements vary with the same sample of blood – March 2014 has a chart

Is the true value 48 nmol -1090 tests @ /is.gd/vitd1090

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
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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.



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