Development and multisite evaluation of an automated assay for B12 on the Abbott AxSYM analyzer.

mutagenic primers are used in this method, it is important to note that they are equally mismatched to both the wild-type and mutant alleles. The specificity is introduced by extension of the primers, which introduces a different base at the first step. This is an important feature of the assay design, removing any possibility of allele-specific amplification, and produces a robust procedure. This technique provides an improved method of detection for both the C282Y and H63D mutations and diagnosis of hemochromatosis. By testing simultaneously for both mutations, this method decreases both the cost and time involved for each assay. The PCR is rapid and yields a consistently high-quality product. This assay is appropriate as a diagnostic test because results can be obtained quickly and confidently at a low cost, making it suitable for introduction into routine clinical laboratories. novel MHC class 1-like gene is mutated in patients with hereditary heamo-The hemochromatosis gene product complexes with the transferrin receptor and lowers its affinity for ligand binding. al. The significance of haemochromatosis gene mutations in the general population: implications for screening. Gut 1998;43:830 – 6. 13. Lynas C. A cheaper and more rapid polymerase chain reaction-restriction fragment length polymorphism method for the detection of the HLA-H gene mutations occurring in hereditary hemochromatosis [Letter]. Reliable measurement of serum B 12 is important in the differential diagnosis of megaloblastic anemia (1). Numerous assays, many of which are manual isotopic methods , have been developed for quantifying B 12. 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 B 12 in human plasma and serum with the Abbott AxSYM random access analyzer (2) that improves on existing B 12 assays. During the assay, sample is first mixed with denaturant and extractant reagents. The denaturant reagent contains NaOH to unfold endogenous B 12-binding proteins to release B 12 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 B 12 (including cobinamide), whereas TC-II and R-protein bind both physiologically inactive B 12 and physiologically active forms (e.g., cyano-cobalamin). 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 B 12 …