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Technical Briefs |
a address for correspondence: University Hospital of Turku, Kiinamyllynkatu 4-8, FIN-20520 Turku, Finland
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) x 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) x
EIA - (1.3994 ± 0.332); Sy|x = 0.28;
r = 0.9877. The equation for serum was: IMx =
(1.0597 ± 0.080) x 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) x 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) x serum + (0.1107 ± 0.832);
Sy|x = 0.67; r = 0.9153; n =
16 individuals.
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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.
Acknowledgments
I thank Aila Sjöholm and Anja Ilmanen for technical assistance and Abbott Diagnostics Division for providing the IMx Homocysteine assay kits for this study.
Footnotes
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{at}tyks.fi
References
The following articles in journals at HighWire Press have cited this article:
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P. Pernet, E. Lasnier, and M. Vaubourdolle Evaluation of the AxSYM Homocysteine Assay and Comparison with the IMx Homocysteine Assay Clin. Chem., September 1, 2000; 46(9): 1440 - 1441. [Full Text] [PDF]
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E. Nexo, F. Engbaek, P. M. Ueland, C. Westby, P. O'Gorman, C. Johnston, B. F. Kase, A. B. Guttormsen, I. Alfheim, J. McPartlin, et al. Evaluation of Novel Assays in Clinical Chemistry: Quantification of Plasma Total Homocysteine Clin. Chem., August 1, 2000; 46(8): 1150 - 1156. [Abstract] [Full Text] [PDF]
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