Analytical and clinical validation of the 25 OH vitamin D assay for the LIAISON® automated analyzer
Introduction
Low vitamin D status is being associated with a growing number of diseases. Severe vitamin D deficiency has long been associated with rickets in children and osteomalacia in adults [1], [2]. Serum concentrations of 25 OH vitamin D of less than 40–50 nmol/l (16–20 μg/l) can lead to secondary hyperparathyroidism and its deleterious consequences on bone mass over the long term [3], [4]. Serum 25 OH Vitamin D concentrations less than 80 nmol/L have been shown to impair absorption of intestinal calcium [5]. Vitamin D deficiency has been associated with disturbed muscle metabolism [6], particularly impaired intracellular calcium metabolism [7]. High circulating concentrations of NT-proANP, a marker of the severity of congestive heart failure, has been associated with low circulating 25 OH vitamin D concentrations [8]. In addition, vitamin D status has been linked to prostate, breast, and colon cancer [9], [10], [11]. Currently, the circulating 25 OH vitamin D concentration is the best available index of vitamin D nutritional status [12], [13]. Therefore, the measurement of 25 OH vitamin D is becoming increasingly important to the diagnosis and management of numerous diseases.
The measurement of 25 OH vitamin D has evolved through several technologies. Several high-performance liquid chromatography (HPLC) methods have been described that are labor intensive, often technique dependent, require costly equipment and large sample volumes [14], [15], [16], [17]. Many assays were developed utilizing the naturally occurring vitamin D binding protein from various species and requiring extraction or chromatographic purification as pretreatment steps [18], [19], [20], [21]. These assays often suffered from significant interferences and cross-reactivities with endogenous vitamin D metabolites. Binding protein assays were generally replaced by radioimmunoassays (RIAs) utilizing either 3H- or 125I-labeled tracers [22], [23]. Recently, automated chemiluminescence-based assays have become available, utilizing either the vitamin D binding protein [24], or a specific antibody [25] in the measurement of serum 25 OH vitamin D concentrations. These assays provide the potential to make vitamin D assessment more accessible to the general practitioner. Validation of assay methodologies is mandatory for the modern clinical laboratory. The present study is designed to provide a baseline for assay validation studies for the LIAISON® 25 OH Vitamin D assay [25].
Section snippets
Analytical and clinical samples
Analytical and clinical samples were obtained from normal healthy adult volunteers or hospital outpatients after informed consent or as repository samples from reference laboratories. Samples were analyzed at three separate sites. The two clinical sites utilized one instrument each, while the analytical site utilized four instruments.
Vitamin D assays
Vitamin D was determined in samples with either of two commercially available 25 OH Vitamin D assays. A radioimmunoassay (DiaSorin, Stillwater, MN) was performed
Assay validation
The minimum detectable concentration, defined as the concentration at two standard deviations from the mean zero standard signal, was determined for three lots. The values determined for each of the three lots were 4.3, 4.3 and 4.5 nmol/L, respectively (data not shown). Therefore, the minimum detectable concentration of the assay was less than 5.0 nmol/L.
Since the minimum detectable concentration was less than the lowest reportable limit of the LIAISON® assay, a functional sensitivity was
Discussion
Analytical and clinical method validation is an important requirement as the methods of assessment of 25 OH vitamin D move from labor and equipment intensive, manual extraction methods in the specialty laboratory to the automated non-extraction methods in the general clinical laboratory. Often the laboratory is provided with only minimal or selective comparison data. The data presented here are intended to provide a rigorous baseline for the clinical laboratory in the evaluation of validation
Acknowledgements
Funding for this work was provided by DiaSorin Inc., Stillwater, MN.
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