Analytical methods and performance of the immunoassay methods for determination of vitamin d in comparison to mass spectrometry / Analitičke metode i izvođenje imunometrijskih određivanja vitamina d u poređenju sa masenom spektrometrijom

Abstract

Summary: Demand for vitamin D testing has been on a constant rise worldwide, partially due to mounting evidence linking vitamin D status to overall health and well-being. Currently available assays measure 25-hydroxy vitamin D (25-OHD), a major circulating form of vitamin D. Available methodologies include immunoassays and mass spectrometry based methods (LC-MS/MS). Until recently, the only immunoassays available for diagnostic use in the US have been DiaSorin radioimmunoassay (RIA) and an automated immunoassay on a LIAISON ® platform. Within the last year, Siemens and Abbott successfully launched immunoassays for determination of total vitamin D on their respective automated platforms, Centaur ® and ARCHITECT ® . Development of robust and precise Vitamin D immunoassays has historically been plagued with difficulty. One of the major challenges is development of specific antibodies against such a small antigen. Vitamin D is also highly hydrophobic molecule predominantly bound to vitamin D binding protein (DBP). It is likely, therefore, that immunoassays might be affected to varying extent by the DBP concentration. Adoption of LC-MS/MS into clinical laboratories has enabled development of accurate and almost fully automated methods that could handle increasing volume demands, especially in large volume reference laboratories. Smaller to mid-size hospital laboratories as well as physician offices have neither funds nor technical expertise to implement LC-MS/MS based testing. Our laboratory at the University of Chicago Medical Center has also seen increase in vitamin D volume and currently performs close to 20,000 25-OHD assays per year. We have recently developed an LC-MS/MS method for quantitation of 25-OHD 2 (obtained from plant sources) and 25-OHD 3 (endogenous and animal sources). Prior to acquisition of LC-MS/MS Kratak sadr`aj: Broj zahteva za odre|ivanjem vitamina D je u konstantnom porastu {irom sveta, delom zbog sve vi{e dokaza koji povezuju status vitamina D sa op{tim zdravljem. Trenutno raspolo`ivim testovima odre|uje se 25-hidroksi vitamin D (25-OHD), glavni oblik vitamina D u cirkulaciji. Po stoje}e metodologije uklju~uju imunometrijska odre |i vanja i tehnike zasnovane na masenoj spektrometriji (LC-MS/MS). Do nedavno, jedine raspolo`ive imunometrijske metode kori{}ene za dijagnostiku u SAD su bile DiaSorin radioimunoodre|ivanje (RIA) i automatizovano imuno odre|ivanje na LIAISON ® platformi. U toku pro{le godine Siemens i Abbott su uspe{no lansirali imunometrijske testove za odre|ivanje ukupnog vitamina D na svojim odgova raju}im automatizovanim platformama, Centaur ® i ARCHI-TECT ® . Razvoj robusnih i preciznih imunometrijskih testova za odre|ivanje vitamina D su, istorijski gledano, pratili problemi. Jedan od najve}ih izazova je razvoj specifi~nih antitela protiv malog antigena. Vitamin D je tako|e jako hidrofoban molekul predominantno vezan za vitamin D vezu ju}i protein (DBP). Stoga postoji verovatno}a da na imuno odre|ivanja u razli~itom stepenu mo`e da uti~e koncentracija DBP. Uvo|enje LC-MS/MS u...

Current trends in the analytical determination of vitamin D

“…In addition, many laboratories rely on commercial immunoassay kits, which have their own pretreatment protocol that differs between laboratories. All of this causes sensitivity and specificity to fluctuate since many factors must be controlled (28,29). Otherwise, even when a good sample treatment is carried out, as well as an appropriate analytical method of vitamin D is chosen, vitamin D levels can be increased or decreased in the same person who maintains similar eating habits and of exposure to the sun during the year.…”

“…The quantification of vitamin D found in biological samples presents recognized difficulties (5). This circumstance coincides with an increase in studies to evaluate the content of vitamin D (6,7) occurring over the last decade, which has been attributed to two factors: on the one hand, the prevalence of serious deficiencies of this vitamin in poor countries and, on the other, an increase in the use of vitamin D as a general marker of health status and because of its relationship with various pathologies (8) Therefore, the need for quality research is paramount due to the aforementioned aspects, which cannot be avoided (23). The problem with immunoassays in the preparation of samples is that an antibody that is so specific to such a small antigen must be determined very clearly, in addition to the hydrophobicity of the vitamin and its binding protein (DBP), which would make oscillations in the amount of DBP present in the sample affect the immunoassay.…”

“…In contrast, very large clinical laboratories and academic institutions use LC-MS/MS methods given that their sensitivity and specificity mean that they are used in those at-risk populations where the accuracy of the method may be very important, such as in the pediatric population, for example. Along the same line, the method can also be chosen depending on the metabolite that you aim to determine, using a more specific test that determines the last precursor of vitamin D in pathologies in which you want to precisely analyze this vitamin such as rickets(28,29).…”