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Reliable measurement of serum B12 is important in the differential diagnosis of megaloblastic anemia (1). Numerous assays, many of which are manual isotopic methods, have been developed for quantifying B12. The shortcomings among the more recently developed automated methods include a need for frequent calibration, a lack of random access capability, complicated reagent preparations, and slow turnaround time. We have developed an automated, nonisotopic assay for measuring B12 in human plasma and serum with the Abbott AxSYM random access analyzer (2) that improves on existing B12 assays.During the assay, sample is first mixed with denaturant and extractant reagents. The denaturant reagent contains NaOH to unfold endogenous B12-binding proteins to release B12 for assay. These endogenous binding proteins include intrinsic factor (IF), transcobalamin-II (TC-II), and R-protein. IF exhibits almost no affinity for physiologically inactive forms of B12 (including cobinamide), whereas TC-II and R-protein bind both physiologically inactive B12 and physiologically active forms (e.g., cyanocobalamin). The extractant reagents contain an excess of cobinamide and cyanide. The cobinamide occupies TC-II and R-protein binding sites to facilitate extraction of the B12, and the cyanide converts the unstable physiological forms of B12 (methylcobalamin, hydroxycobalamin, and adenosylcobalamin) into the stable cyanocobalamin form. After the extraction step, microparticles coated …

Development and Multisite Evaluation of an Automated Assay for B12 on the Abbott AxSYM Analyzer