Approaches to measurement of Vitamin D concentrations - Immunoassays

Abstract

The clinical relevance of vitamin D calls for analytically reliable and cost-effective testing methods. 25-hydroxyvitamin D (25(OH)D), the storage form of vitamin D in the blood circulation, is widely accepted as the best indicator of the individual vitamin D status. 25(OH)D immunoassays play a major role in routine testing in the clinical laboratory and many new automated immunoassays have been introduced to the market by the diagnostic industry in recent years. Detectability, precision, traceability and comparability to the reference method liquid chromatography – tandem mass spectroscopy (LCTMS) are essential quality requirements for 25(OH)D immunoassays. The hydrophobic nature of the analyte, the high concentration and affinity of vitamin D binding protein (VDBP) in serum and the cross-reactivity requirements due to the broad spectrum of metabolites of vitamin D are most demanding for assay development. It requires an adequate assay design including a thorough pretreatment process to inactivate the VDBP, careful selection of the specifier (antibody or binding protein) to meet the cross-reactivity requirement, and standardization of the assay versus LCTMS to achieve comparability of results between methods.

Key Words::

• 25-Hydroxyvitamin D
• Vitamin D binding protein
• VDBP

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Introduction

The association between low vitamin D status and increased risk for several diseases, in particular osteoporosis, has received extensive attention in recent years and has driven an ever increasing demand for measuring vitamin D concentrations in serum or plasma. Routine testing of vitamin D status in the clinical laboratories calls for analytically reliable and cost-effective methods. Immunoassays are suited for this purpose, because they can be sensitive and specific enough, easily automated and integrated in the routine laboratory organization.

The 25-hydroxyvitamin D (25(OH)D concentration as the sum of 25-hydroxyvitamin D3 (25(OH)D3) and 25-hydroxyvitamin D2 (25(OH)D2) concentrations) concentration is widely accepted as the best indicator of the vitamin D status of an individual. It covers not only the endocrine but also the paracrine biological pathways of vitamin D, whereas the active hormone 1,25-di-dydroxyvitamin D (1,25(OH)₂D) does not provide information on the vitamin D status and is often normal or increased as the result of secondary hyperparathyroidism associated with vitamin D deficiency [1]. The requirements for those two sets of analytes are quite different and need different assay design. In consideration of its wide spread use and importance in clinical practice, this paper will focus solely on the measurement of 25(OH)D concentration.

Requirements for measurement of 25(OH)D concentrations

Recognition of metabolites

Although the utility of vitamin D2 supplementation is under debate, a 25(OH)D assay should detect 25(OH)D2 as well as 25(OH)D3. A multitude of vitamin D metabolites have been described in literature, but the information on their clinical relevance and concentration in serum is only limited. Many of these components are constitutional or stereo-isomers and may also be detected in MS methods. The effect of metabolites on the test result should be minimized.

Traceability and comparability to reference methods

Immunoassays should be calibrated against liquid chromatography-tandem mass spectroscopy (LC-TMS), which is considered as a reference method for measuring 25(OH)D concentrations in serum or plasma and should give comparable results to allow traceability and implementation of general clinical guidance for vitamin D supplementation.

Detection limit and precision

There is a broad consensus that the 25(OH)D concentration in serum should be between 50–80 nmol/L for sustained bone health [2]. A more recent consensus of experts leads to the conclusion that for general health a 25(OH)D concentration of ≥–75 nmol/L is desired [3]. To allow clinical decision making the limit of quantification (LoQ) for any 25(OH)D assay should be below 25 nmol/L and intermediate imprecision according to CLSI guideline EP5 should be well below 10 % in the concentration interval between 50 and 150 nmol/L.

Automation

Ready for use reagents and fully automation of all process steps is desired to allow a cost efficient high volume measurement of 25(OH)D concentration.

Design of immunoassays

25(OH)D immunoassays are designed in a competitive assay format: A 25(OH)D binding component (specifier) is mixed with a 25(OH)D-conjugate, forming a complex during incubation. At the end of the incubation time the complex is separated from the reaction mixture and detected. The specifier may be an antibody against 25(OH)D or a vitamin D binding protein (VDBP). The 25(OH)D-conjugate may carry the label for signal generation or a tag for binding to a solid phase to enable separation of the complex. When the sample is added to the reagent mixture, 25(OH)D of the serum sample competes with the 25(OH)D-conjugate for binding to the specifier, hence with increasing 25(OH)D concentration the signal decreases and the calibration curve shows a negative slope.

The required LoQ of <–25 nmol/L for 25(OH)D measurement procedures is not a particular challenge for immunoassay technologies, however, the very hydrophobic nature of the analyte, the high concentration of VDBP in the sample and the cross-reactivity requirements due to the broad spectrum of metabolites and variants are most demanding for assay development. VDBP is the main transport protein of 25(OH)D in the blood circulation [4]. VDBP binds with similar high affinity to 25(OH)D3 and 25(OH)D2, less than 1 % of the circulating 25(OH)D is free [5,6]. Moreover, VDBP is found in serum with a median concentration of approximately 8 μmol/L and may vary at least between 4 and 14 μmol/L [7]. VDBP concentrations exceed the concentration of the analyte in the clinical decision interval by a factor of about 100–1000. To allow detection by immunoassay the 25(OH)D must be quantitatively released from the binding protein, the binding protein itself must be thoroughly separated from the sample or completely inactivated. Residual active binding protein of as little as 4 nmol/L (approx. 0.5‰ of the total VDBP) may interfere with the measurement. If the releasing step is not effective, one will see differences in 25(OH)D recovery between an immunoassay and LC-TMS reference method depending on the VDBP concentration in the sample.

Most immunoassays use a pretreatment procedure to release 25(OH) D and remove or inactivate the Vitamin D binding protein. Manual methods use acetonitrile precipitation with subsequent centrifugation for the separation of VDBP. Precipitation and centrifugation cannot be easily automated, hence manufacturers opt for release and inactivation by adding combinations of organic solvents like ethanol or methanol, alkaline denaturation and releasing substances like 8-anilino-1-naphtalensulfonic acid (ANSA) to the sample. The pretreated sample can then further be processed in the competitive immunoassay format as described above. An overview of pretreatment solutions used in commercially available 25(OH)D assays is given in Table I.

Table I. Serum 25(OH)D concentrations and their interpretation.

Classification	Serum 25(OH)D nmol/L (ng/mL)	Clinical implications
Vitamin D deficiency	<50 nmol/L (<20 ng/mL)	Summarizes both concentrations of severe deficiency and insufficiency
Severe vitamin D deficiency	<25 nmol/L (<10 ng/mL)	Increased risk of rickets, osteomalacia, secondary hyperpara-thyroidism, myopathy, falls, fractures
Vitamin D insufficiency	25 to 49 nmol/L (10 to 19 ng/mL)	Increased risk of bone loss, secondary hyperparathyroidism, falls, fractures
Adequate vitamin D threshold concentration*	50 nmol/L (20 ng/mL)	Low risk for bone loss and secondary hyperparathyroidism, neutral effect on falls and fractures
Desirable vitamin D threshold concentration for fall and fracture reduction**	75 nmol/L (30 ng/mL)	Optimal suppression of parathyroid hormone and bone loss; reduction of falls and fractures by about 20 %

*Threshold supported by the Institute of Medicine as adequate concentration for most people (97 %) [17].

**Threshold supported by the IOF [18] and US Endocrine Society [19] for the reduction of falls and fractures, especially among older adults age 60+ years of age.

Testing sera of pregnant women is a good litmus test for the effectiveness of the pre-treatment procedure, because they show elevated concentrations of VDBP [8,9,10]. Many current immunoassays show a different recovery vs. LC-MS in sera of pregnant women compared to healthy individuals [11].

Another critical factor for the comparability to the reference method is the detection of metabolites and variants. In an immunoassay, the spectrum of metabolites or variants detected is mostly determined by the cross reactivity pattern of the antibody or binding protein used. The current immunoassays are either based on polyclonal or monoclonal antibodies or human recombinant VDBP (see Table I). All of them show cross-reactivity (XR) to metabolites. The impact of a particular metabolite on the test result can be calculated by (XR(%) × concentration of metabolite). If this term is small compared to the analyte concentration, it can be neglected. Moreover, if the concentration of the metabolite correlates with 25(OH)D concentrations, the systematic bias can be corrected by an adequate standardization of the assay.

More than forty metabolites have been identified [13,14], but our knowledge of the clinical relevance of Vitamin D metabolites is still limited and data on their serum concentrations is scarce. Moreover, many of the metabolites are not available as pure substances for XR-testing. Manufacturers of automated immunoassays typically disclose cross-reactivity data for 1,25(OH)₂D, vitamin D2, vitamin D3, 24,25-dihydroxyvitamin-D3 (24,25(OH)₂D3 and 25(OH)-3-Epi-vitamin D3 (3Epi 25(OH)D3) in their package inserts. While the impact of the first ones is considered to be small, because either the concentration of the metabolite or the XR of the assay is very low, 24,25(OH)₂D3 and 3Epi 25(OH)D concentrations have to be discussed in more detail. 24,25(OH)₂D3 concentrations in serum is in the interval of 10–15 % of 25(OH)D3, correlates well with 25(OH)D3 and is only modestly affected by vitamin D supplementation [15]. The average bias due to 24,25(OH)₂D3 can be minimized by reference calibration using human samples with known metabolite content established by LC-Tandem-MS. The average serum concentration in adults of 3Epi 25(OH)D is approx. 5 times lower than 25,25(OH)₂D3 and the overall impact on the test result very low. In addition, there are reports that the C3-epimerization pathway of vitamin D produces biologically active 1,25-dihydroxy-3-epi-vitamin D3 exhibiting similar properties as 1,25(OH)₂D3 [16]. As a consequence one may consider 3Epi 25(OH)D as a part of the “total” 25-OH-Vitamin D on an equal footing with 25(OH)D3 and 25(OH)D2. Finally, it has also to be taken into account that routine LC-TMS methods may also detect constitutional and stereoisomers of 25(OH)D with impact on comparability to immunoassays.

Future of immunoassay testing

The quality and degree of automation of immunoassays did significantly improve in the last years. Many new assays for 25(OH)D have been brought to the market on immune assay testing platforms which can be easily integrated into laboratory routine. There is a clear tendency to calibrate commercial assays against LC-TMS (Table II) and many assays show already an acceptable comparability in cohorts of healthy individuals [11], i.e. between 0.9 and 1.1 and correlation coefficients of 0.85–0.95 in method comparisons.

Currently available automated immunoassays cover the clinically relevant measuring interval (Table II) and show cross-reactivity to 25(OH)D2 between 50–100 % according to the package insert information.

A lot has been done, but there is still a lot to do to lift the state of the art of 25(OH)D concentration measurements to the level of other steroid hormone tests. The improvement should focus on the comparability to LC-TMS overall as well as in individual samples. Binding protein inactivation and the recognition of metabolites are key factors for this assay feature. While adequate pretreatment procedures are available already today, as we could see from the data in sera of pregnant women, the improvement of metabolite recognition will need further research. First and foremost, our knowledge on vitamin D metabolites has to be extended to get the full picture of metabolite concentrations in human sera and the clinical significance thereof. Based on this knowledge one may look for better specifiers – antibodies or binding proteins – to tailor the cross-reactivity to the various components to reflect best the Vitamin D status of an individual.

Questions and answers

R Vieth, Canada

A physician would want to know how the concentration of the analyte relates to something clinical. In the future I would like to see which method best predicts a clinical outcome. It is well known that vitamin D is related to PTH and if I were a physician, I would like to choose the method which best predicts PTH.

J Billen, Belgium

The main clinical use of the assay is to diagnose vitamin D deficiency. You aim at a detectability of 25 nmol/L. If you can get down to 1–2 nmol/L with LCMS method, and if you know that patients can have levels between 1 and 25 nmol/L, have you been ambitious enough in the sensitivity you can achieve?

B Ofenloch-Haehnle

The issue is what we want to achieve with the assay. If the detectability can be improved, it may be useful but the major intended use, and thus optimisation, of assays is to diagnose deficiency. I think good trueness and precision in that interval is therefore the priority. If other elements, such as higher detectability can be achieved, that is a bonus. Some assays already claim a detectability of 7.5 nmol/L.

G Jones, Canada

You have mentioned that 24,25(OH)₂D3 is a problem and in most samples it is a small problem, but in toxicity, there are lots of other metabolites present which will almost certainly inflate the result. Do you have an upper limit above which you don't recommend the assay? How accurate is the assay at high concentrations?

B Ofenloch-Haehnle

This is more a clinical question. What is the threshold you would want to measure?

G Jones

Probably over 250 nmol/L. Are you confident you can measure that accurately?

B Ofenloch-Haehnle

Yes, with an additional dilution step.

Declaration of interest: The author report no conflicts of interest. The author alone is responsible for the content and writing of the paper.