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

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 immuno - assays 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 hy - drophobic molecule predominantly bound to vitamin D binding protein (DBP). It is likely, therefore, that immuno - assays 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 in - crease in vitamin D volume and currently performs close to 20,000 25-OHD assays per year. We have recently deve - loped an LC-MS/MS method for quantitation of 25-OHD2 (obtained from plant sources) and 25-OHD3 (endogenous and animal sources). Prior to acquisition of LC-MS/MS instrument, we performed 25-OHD analysis by RIA. Du - ring the transition period, we encountered several challenges, including the necessity to streamline sample preparation as well as the bias introduced by calibration dif ferences. We chose to match our LC-MS/MS method to the RIA method in order to make this transition transparent to the clinician. Most immunoassays available today are acceptable for clinical use and might be method of choice for smaller laboratories. Larger clinical laboratories and aca demic institutions that possess technical expertise, particularly the ones with large pediatric population where assay sensitivity and specificity may be important, might find LC-MS/MS methodology a more suitable choice. Kratak sadržaj 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 va - nja i tehnike zasnovane na masenoj spektrometriji (LCMS/ 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 Sie - mens i Abbott su uspe{no lansirali imunometrijske testove za odre|ivanje ukupnog vitamina D na svojim odgova ra - ju}im automatizovanim platformama, Centaur® i ARCHITECT ®. 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 klini~ke laboratorije je omogu}ilo razvoj ta~nih i skoro potpuno automatizovanih metoda koje bi mogle da obrade rastu}i broj zahteva za analizu, naro~ito u referentnim laboratorijama sa velikim obimom posla. Klini~ke laboratorije manjeg do srednjeg obima, kao i lekarske ordinacije, ne raspola`u finansijskim sredstvima niti tehni~kim znanjem za implementaciju odre - |ivanja zasnovanog na LC-MS/MS. U na{oj laboratoriji u Medicinskom Centru Univerziteta u ^ikagu je tako|e pri - me }en porast broja zahteva za odre|ivanje vitamina D i tre - nutno se izvr{i blizu 20 000 odre|ivanja 25-OHD go di{nje. Nedavno smo razvili LC-MS/MS metodu za kvantifikaciju 25-OHD2 (dobijen iz biljnih izvora) i 25-OHD3 (iz endogenih i `ivotinjskih izvora). Pre nabavke LC-MS/MS aparata analiza 25-OHD je ra|ena RIA metodom. U pre laznom periodu, nai{li smo na nekoliko izazova, uklju~uju}i neo - phodnost jednostavnije pripreme uzorka, kao i odstupa nje nastalo razlikama u kalibraciji. Odlu~ili smo da upore dimo na{u LC-MS/MS metodu sa RIA metodom da bi ovaj prelaz postao transparentan za klini~are. Ve}ina imuno odre |i va - nja koja su danas na raspolaganju su prihvatljiva za klini~ku upotrebu i mogu biti metode izbora za manje la boratorije. Za ve}e klini~ke laboratorije i akademske institucije koje poseduju tenhni~ku obu~enost, naro~ito one sa velikom pe dijatrijskom populacijom gde osetljivost i specifi~nost mogu biti va`ne, LC-MS/MS metodologija mo`e biti adekvatniji izbor.