Fully Automated Measurement of Total Homocysteine in Plasma and Serum on the Abbott IMx Analyzer

Increasing evidence indicates that high total homocysteine (tHcy) may be causally related to several clinical situations, such as cardiovascular disease, birth defects, and folate and vitamin B12 deficiency (1)(2)(3)(4). Accordingly, the interest in tHcy determinations in blood has increased in routine and research laboratories. Several methods, such as HPLC, stable isotope dilution, and enzyme immunoassay, have been used for the determination of tHcy, but they are time-consuming and require highly skilled technical staff. Recently, an automated tHcy assay has come on the market. This IMx Homocysteine assay (Abbott Laboratories) is a fluorescence polarization immunoassay for the quantitative measurement of tHcy in human plasma and serum with no manual sample pretreatment (5). For the evaluation of the method, EDTA plasma and serum samples were collected simultaneously from 32 men, ages, 45-67 years. After collection, the samples were kept on ice, centrifuged within 1 h, and stored at −70 °C until analysis. The analytical performance of the automated IMx Homocysteine assay was compared with the manual Hcy enzyme immunoassay kit (Axis EIA Homocysteine; Axis Biochemicals) on microtiter plates with spectrophotometric measurement of peroxidase activity (6). Both assays are based on enzymatic conversion of Hcy to S-adenosyl-l-homocysteine (SAH) by the action of SAH hydrolase (SAHase; EC 3.3.1.1), followed by quantification of SAH in a competitive immunoassay with the use of a monoclonal anti-SAH antibody. The comparison of the IMx Homocysteine assay with the Axis EIA Homocysteine assay yielded the following equation: IMx = (1.0753 ± 0.042) × EIA − (1.2949 ± 0.459); Sy|x = 0.54; r = 0.955; n = plasma and serum samples from 16 individuals (Fig. 1 A). When plasma and serum samples were considered separately, the equation for plasma was: IMx = (1.0738 ± 0.032) × EIA − (1.3994 ± 0.332); Sy|x = 0.28; r = 0.9877. The equation for serum was: IMx = (1.0597 ± 0.080) × EIA − (1.0057 ± 0.885); Sy|x = 0.71; r = 0.9261. The mean (± SD) values for plasma and serum samples were 11.3 (± 3.58) and 12.4 (± 3.76) μmol/L on the IMx and 10.1 (± 2.23) and 10.8 (± 2.30) μmol/L on the EIA, respectively. The parallel measurement of plasma and serum Hcy with IMx Homocysteine assay gave the equation: plasma = (0.9571 ± 0.010) × serum − (0.5163 ± 0.147); Sy|x = 0.34; r = 0.997; n = 32 individuals (Fig. 1B). Correspondingly, the equation with Axis EIA Homocysteine assay was: plasma = (0.9245 ± 0.008) × serum + (0.1107 ± 0.832); Sy|x = 0.67; r = 0.9153; n = 16 individuals. Correlation between tHcy determined by the IMx Homocysteine assay and by the Axis EIA Homocysteine assay (A), and comparison between plasma and serum samples determined with the IMx Homocysteine assay (B). (A), the equation for the line is: y = 1.0753x − 1.2949; r2 = 0.9547; n = plasma and serum samples from 16 individuals. (B), the equation for the line is: y = 0.9571x − 0.5163; r2 = 0.9968; n = plasma and serum samples from 32 individuals [including samples used in panel A]. The imprecision of the IMx Homocysteine assay was evaluated using NCCLS guidelines (7). Two IMx assays were performed on 5 days with serum controls of 7.0, 12.5, and 25.0 μmol/L Hcy assayed in replicates of two. The within-assay CVs were 1.8%, 1.9%, and 1.3%; the between-assay CVs were 0.8%, 0.3%, and 0.7%; and the total CVs were 1.9%, 1.8%, and 1.4%, respectively. The detection limit for the assay was 0.44 μmol/L, which was measured by assaying the zero calibrator in replicates of 10 in four IMx assays and calculated as 3 SD of the mean response of the zero calibrator (0.165 ± 0.09). The sample volume requirement was 50 μL, the stated measuring range was 0.5–50 μmol/L, and the throughput was 20 samples per hour. The observed results indicate that the IMx Homocysteine assay provides a precise and easy-to-use quantitative measurement of tHcy in plasma and serum for routine use. The automation of cumbersome manual steps in currently used methods of Hcy measurement may reduce analytical variability between studies. Research and Development Centre of Social Insurance Institution and Department of Clinical Chemistry, Central Laboratory, University Hospital, Turku, Finland fax 358-2-2613920, e-mail aila.leino@tyks.fi I thank Aila Sjöholm and Anja Ilmanen for technical assistance and Abbott Diagnostics Division for providing the IMx Homocysteine assay kits for this study.