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Vitamin D Testing Methods

Vitamin D Testing

Vitamin D testing has significantly increased in recent years, most likely due to emerging evidence linking deficiency to the development of cancer, diabetes, autoimmune and cardiovascular diseases.
As a result, increased demands have led to more commercially available automated vitamin D assays. As recent as 2012, in an effort to meet demands, several manufacturers have had proficiency issues and are working on re-standardizing their Vitamin D assay to produce more reliable results.

The National Institutes of Health, Office of Dietary Supplements established the Vitamin D Standardization Program in November 2010 to standardize the laboratory measurement of vitamin D status worldwide. Standardization is essential to improve the detection, evaluation, and treatment of vitamin D deficiency and insufficiency by making measurements of serum total 25-hydroxyvitamin D [25(OH)D] accurate and comparable over time, location, and laboratory procedure.

Contact our friendly and helpful staff at (855) 765-7767 to discuss an accurate, cost effective, and reliable Vitamin D testing method for practice today!

Who Should Be Tested?

Deficiencies at any stage of life can have devastating consequences. According to Mayo Clinic, the following patients should be screened for Vitamin D deficiency:

  • Individuals receiving therapy to prevent or treat osteoporosis
  • Elderly people, especially those with minimal exposure to sunlight
  • Patients with signs and symptoms of hypocalcemia or hypercalcemia
  • Children and adults with suspected rickets and osteomalacia, respectively
  • Patients receiving vitamin D therapy who do not demonstrate clinical improvement

FDA Approved Vitamin D Manufacturers

Click Manufacturer to Read About Methodology

DiaSorin (LIAISON®) FDA clearance Oct 2007
ESA Bioscience (HPLC) FDA clearance May 2008
Siemens Healthcare Diagnostics (ADVIA) FDA clearance Oct 2011
Abbott Diagnostics (ARCHITECT) FDA clearance Nov 2011
Ortho-Clinical Diagnostics (VITROS®) FDA clearance June 2012
Roche Diagnostics (ELECSYS) FDA clearance July 2012
Tosoh Bioscience (AIA-Pack) FDA clearance Feb 2013

Recent Vitamin D Methods Often Inaccurate

Faster, Less Expensive Tests May Overestimate Vitamin D Deficiency
According to a recent study in 2012 by Mayo Clinic, new lab tests which measure blood levels of Vitamin D are inaccurate 40% of the time. (click here to read entire article)

LIAISON® Vitamin D Testing Method

The LIAISON® 25 OH Vitamin D assay is a direct competitive chemiluminescence immunoassay (CLIA) for quantitative determination of total 25 OH vitamin D in serum. During the first incubation, 25 OH Vitamin D is dissociated from its binding protein and binds to the specific antibody on the solid phase. After 10 minutes the tracer, (vitamin D linked to an isoluminol derivative) is added. After a secondlO0 minute incubation, the unbound material is removed with a wash cycle. Subsequently, the starter reagents are added to initiate a flash chemiluminescent reaction. The light signal is measured by a DiaSorin LIAISON® 25 OH Vitamin 0 TOTAL Premarket Notification photomultiplier as relative lig ht units (RLU) and is inversely proportional to the concentration of 25 OH vitamin D present in calibrators, controls, or samples. Changes were made to the assay’s magnetic particle surface and the Assay Buffer formulation to reduce reaction with rare heterophilic antibodies.

ESA, Inc. Vitamin D Testing Method

The ESA method is a complete kit for measurement of Total 25(OH)D by HPLC with electrochemical (EC) detection. Specific reagents and solid phase extraction columns are included for sample preparation and are employed with user-supplied standard laboratory equipment (centrifuge, test tubes, pipettes,. etc.). A 2001.L volume of sample (serum or plasma) is mixed with a precipitation reagent, which contains internal standard (IS).

The internal standard is a stable vitamin D analogue that is used to correct for variability in extraction recovery and analytical sample volume. After centrifugation, supernatant is poured onto a pre-conditioned SPE column for rapid extraction of 25(OH)D and IS. SPE columns are washed with 2 different reagents and analytes are eluted with a third reagent. The resulting eluent is diluted before analysis. The prepared sample is analyzed with an isocratic HPLC system using an ESA EC detector (Coulochem® III or CoulArray®) equipped with a dual coulometric EC cell. Calibration is accomplished by direct HPLC analysis of authentic standard solutions (i.e. not taken through the extraction step). Analysis requires a specific guard and analytical column, mobile phase and calibration reagents to allow rapid quantitative analysis.

A dual EC cell is used with the first, upstream, cell maintained at a specific potential to oxidatively screen possible interfering sample components. The second, downstream cell is maintained at a potential that is optimized for selective 25(OH)D detection. The dual coulometric EC cell is a rugged detector that provides much higher selectivity and sensitivity than commonly used absorbance detectors. This allows the use of lower sample volumes than are typically required with HPLC-UV methods and is less susceptible to interferences. Analytical run time is less than 12 minutes and Total 25(OH)D sample concentration is automatically determined by single-point internal standard quantitation.

ADVIA Centaur Vitamin D Testing Method

The ADVIA Centaur Vitamin D Total assay is a one-pass 18 minute competitive immunoassay that uses an anti-fluorescein labeled (FITC) monoclonal antibody covalently bound to paramagnetic particles (PMP), one monoclonal antibody labeled with acridium ester (AE), and a Vitamin D analog labeled with fluorescein. An inverse relationship exists between the amount of Vitamin 0 present in the patient sample and the amount of relative light units (RLUs) detected by the system.

ARCHITECT Vitamin D Testing Method

The ARCHITECT 25-OH Vitamin 0 assay is a delayed one-step immunoassay including a sample pre-treatment for the quantitative determination of vitamin D in human serum and plasma using CMIA technology with flexible assay protocols, referred to as Chemiflex.

Sample and pre-treatment reagent are combined. An aliquot of the pre-treated sample is combined with assay diluent and paramagnetic anti-vitamin D coated microparticles to create a reaction mixture. Vitamin D present in the sample binds to anti-vitamin D coated microparticles. After incubation a biotinylated vitamin D anti-Biotin acrid in iu m-labeled conjugate complex is added to the reaction mixture and binds to unoccupied binding sites of the anti-vitamin 0 coated microparticles. After washing, pre-trigger and trigger solutions are added to the reaction mixture. The resulting chemiluminescent reaction is measured as relative light units (RLUs). An indirect relationship exists between the amount of vitamin D in the sample and the RLUs detected by the ARCHITECT i System optics.

VITROS Vitamin D Testing Method

The test uses an immunoassay design to measure vitamin D levels in human plasma or serum. Specifically, a patient blood sample, dissociation buffer and conjugate buffer are sequentially added to a VITROS® MicroWell coated with a monoclonal antibody (MAb). Dissociated vitamin D in the patient sample competes with the vitamin D conjugate to bind to the antibody. After an incubation period, unbound materials are removed by a wash. An enhanced chemiluminescence substrate is then added and light emission is measured to determine the sample’s vitamin D levels.

ELECSYS Vitamin D Testing Method

The Elecsys Vitamin D Assay is a competitive protein binding assay which description uses Vitamin D Binding Protein instead of monoclonal antibodies for detection of 25-Hydroxyvitamin D. The total duration of the assay is 27 minutes. The sample is treated with pretreatment reagent in the first incubation period. This releases any vitamin D from the endogenous vitamin D binding protein present in the patient’s sample. In the next incubation, vitamin D binding protein labeled with ruthenium is added and a complex is formed between the vitamin D (25-OH) and the muthenylated vitamin D binding protein. In the 3rd and final incubation, streptavidin-coated microparticles are added along with vitamin D (25-OH) labeled with biotin. Any unbound ruthenium labeled vitamin D binding proteins become occupied with biotin-labeled vitamin D (25-OH). The complex consisting of the ruthenylated vitamin D binding protein and the biotinylated vitamin D (25-OH) becomes bound to the solid phase via interaction of the biotin and streptavidin. The reaction mixture is aspirated into the measuring cell where the electrochemiluminescence emission is detected. Results are determined using a calibration curve that is generated specifically on each instrument by a 2 point calibration provided with the reagent bar code.

Tosoh Vitamin D Testing Method

ST AIA-PACK 25-OH Vitamin D is designed for in vitro diagnostic use only for the quantitative measurement of total 25-hydroxyvitamin D (25-OH vitamin D) in human serum, Na-heparinized or EDTA plasma on TOSOH AIA System Analyzers. The Tosoh ST AIA-PACK 25-OH Vitamin D is intended as an aid in the determination of Vitamin D sufficiency.

The ST AIA-PACK 25-OH Vitamin assay consists of a pretreatment step to release 25-OH Vitamin D from its serum transporter. Pretreated samples are then added to a ST AIA-PACK 25-OH Vitamin D test cup that contain 25-OH vitamin D-specific monoclonal antibody immobilized on magnetic beads to which 25-OH Vitamin D binds. Next, 25-OH vitamin D conjugated to bovine alkaline phosphatase is added to the reaction mixture, which competes with 25-OH vitamin D for binding to the antibody on magnetic beads. Subsequently, the magnetic beads are washed to remove the unbound enzyme labeled 25-OH vitamin D and are then incubated with a fluorogenic substrate, 4-methylumbelliferyl phosphate (4MUP). The amount of enzyme-labeled 25-OH vitamin D that binds to the beads is inversely proportional to the 25-OH vitamin D concentration in the test sample.


1 Singh RJ. Are clinical laboratories prepared for accurate testing of 25-hydroxyvitamin D? Clin Chem 2008; 54:221-223.

2 Hollis BW and Horst RI. The assessment of circulating 25(OH)D and 1,25(OH)2D: where are we and where are we going. J Steriod Biochem Mol Biol 2007; 103:473-476.


  • Parikshit Bali
    Posted February 16, 2015 at 8:31 am | Permalink

    Need information about Vit D testing.
    Can it be programmed on biochemistry Analyse?
    I have Star 21 plus biochemistry analyser from Rapid diagnostic.

  • Posted May 11, 2016 at 7:23 am | Permalink

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