Standardization of 25(OH)D analysis

The measurement of 25-hydroxyvitamin D  [25(OH)D] levels is affected by assay interference and variability. This article describes the steps that are being taken to improve assay standardization.

by Prof. Etienne Cavalier and Dr Jean-Claude Souberielle

The number of 25-hydroxyvitamin D [25(OH)D] determinations has dramatically increased over the last ten years. This increase can be explained by the growing awareness of the medical community (as well as the general population) of the high prevalence of vitamin D deficiency and its potential impact on numerous diseases beyond bone pathologies, such as cardiovascular diseases, autoimmune diseases, diabetes, malignancies, etc. [1]. This increasing number of requests has led most of the clinical laboratories to move from the DiaSorin radioimmunoassay (RIA), the most widely used method in the late 20th and early 21st centuries, to methods presenting a larger throughput, i.e. automated immunoassays or liquid chromatography tandem mass spectrometry (LC-MS/MS). Also, one has to remember that analytical 25(OH)D determination is far from an easy task – several important problems have to be overcome to correctly assess this parameter.

Pratical difficulties
Among them, the very high lipophilic nature of the molecule and its strong association with vitamin D binding protein (VDBP) and, to a lesser extent, albumin necessitates a thorough separation step and, for the one-phase immunoassays, a good equilibrium between the analyte and the antibodies used in the kits [2]. VDBP can be present at different concentrations according to some physiological or pathological conditions, such as race [3] , pregnancy or chronic kidney disease, which could influence the kinetics of 25(OH)D release [4, 5]. Vitamin D can be found as 25-hydroxyvitamin D2 [25(OH)D2] or 25-hydroxyvitamin D3 [25(OH)D3] and an assay should measure both forms [6]. Other different metabolites of vitamin D, i.e. C3-epimer or 24,25-dihydroxyvitamin D3 can be present in the serum of the patients at different levels, possibly interfering with either immunoassays or LC-MS/MS methods [7]. As with any other immunoassays, vitamin D assays are prone to heterophilic antibody interference, leading to potentially spurious results [8]. Last but not least, standardization of the different assays remains a major problem. Indeed, previously, when the DiaSorin RIA was the only test kit available, all the results obtained could be transposable throughout the world – even though analytical skill and inter-laboratory variation can be a problem with such a manual method – and all the clinicians could compare the results obtained in their patients with the cut-offs obtained in the observational or interventional studies that had used this assay. The increased use of chromatographic methods has shown that the results obtained with the DiaSorin RIA were often lower than those obtained with chromatographic methods. These chromatographic methods were most often ‘home-brewed’ methods and were calibrated against a curve obtained by gravimetric addition of known concentrations of 25(OH)D. Unfortunately, the results generated by these methods presented a high variability, mainly due to extraction and calibration problems, and the results obtained by LC-MS/MS could not be compared from one lab to the other.

Assay standardization
Different standards were then issued by the National Institute of Standards and Technology (NIST 972) and LC-MS/MS methods could thus be calibrated against this material. Unfortunately, due to different matrix problems, it was impossible for immunoassays manufacturers to use these standards to calibrate their assays. They thus chose to indirectly calibrate their tests on serum standards whose concentration had been established with a LC-MS/MS method traceable to the NIST 972 or to remain indirectly calibrated against the DiaSorin RIA. To overcome this problem, a worldwide Vitamin D Standardization Program (VDSP), coordinated by the Centers for Disease Control and Prevention (CDC), the NIST and the University of Ghent, is ongoing: 40 serum samples obtained in apparently healthy single donors and containing 25(OH)D amounts determined with a LC-MS/MS method accepted now as the only reference method for 25(OH)D determination are distributed to laboratories and manufacturers and are expected to allow an unambiguous calibration of the commercially available or the ‘home-brewed’ methods, as well as all the immunoassays [9].

Limitations of assay standardization

This ambitious analytical programme will certainly reduce the variation observed between the methods and the laboratories in healthy individuals. Nevertheless, other problems listed below will remain unsolved:

1. ‘Special’ populations
In patients presenting special patterns of the serum, such as pregnant women or hemodialysed patients, the standardization seems to be less efficient [4, 5].

2. 25(OH)D2 standardization and recovery
As none of the VDSP calibration samples contain significant amount of 25(OH)D2, matrix effects have been described with the NIST samples that have been spiked with 25(OH)D2 and cross-reactivity of the immunoassays with 25(OH)D2 is difficult to establish with precision.

3. C3-epimer
The 3-epi-25-hydroxyvitamin D [3-epi-25(OH)D] presents an identical mass/charge ratio as 25(OH)D. This epimer has an unknown physiological role but may be found in some particular conditions, i.e. in neonates, in patients suffering from liver deficiency or in those presenting high levels of 25(OH)D. Even if not extensively studied, the immunoassays do not seem to cross react with the C3-epimer. However, the separation of 25(OH)D3 and 25(OH)D2 from their epimeric forms 3-epi-25(OH)D with LC-MS/MS methods can only be achieved by derivatization, use of chiral or cyano chromatographic columns and longer runs. Unfortunately, many LC-MS/MS methods (commercial or ‘home-brewed’) have not paid attention to the C3-epimer, resulting in an overestimation of 25(OH)D levels. Thus, even if ‘standardized’, those methods will provide inaccurate results.

4. 24,25-dihydroxyvitamin D
The production of 24,25(OH)2D is linked to the activation of the CYP24A1 in the kidney to inactivate vitamin D. This enzyme is tightly regulated, mainly by PTH and FGF23, showing that this pathway is of physiological importance. 24,25(OH)2D can be present at variable concentrations in the serum, but may be up to 10% of 25(OH)D concentrations. Of note is that 24,25(OH)2D concentrations are proportionally higher in the higher range of 25(OH)D. If there is no interference of 24,25(OH)2D with LC-MS/MS methods, a cross-reactivity, of differing extent is observed with immunoassays, particularly at higher concentrations of 25(OH)D. This can thus result in an overestimation of the ‘true’ 25(OH)D value being observed with immunoassays.

5. Impact of re-standardization on clinical cut-offs
 Traditionally, values of 20 or 30 ng/mL are used as clinical cut-offs to define vitamin D sufficiency. These cut-offs derive from studies that generally used the DiaSorin RIA for 25(OH)D measurements. Using these cut-offs with immunoassays or LC-MS/MS methods that are differently calibrated is thus hazardous. Re-standardization will improve the method-to-method variability, but will also consequently impact the value of the cut-offs, that will also need to be updated according to the new standardization.

In conclusion, vitamin D assays need to be standardized, and also improved in terms of accuracy. The VDSP is an ongoing programme that will improve the lab-to-lab consistency, even if different problems will, however, persist.

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The authors
Etienne Cavalier*1 PhD, PharmD, European Specialist in Laboratory Medicine (EuSpLM) and Dr Jean-Claude Souberielle2 PhD, EuSpLM
1 Department of Clinical Chemistry, University of Liège, Liège, Belgium
2 Hôpital Necker-Enfants malades, Paris, France

*Corresponding author