Comparison of three methods for total homocysteine plasma determination
Related Experiment Video
Updated: Oct 5, 2026
Integration of Miniaturized Solid Phase Extraction and LC-MS/MS Detection of 3-Nitrotyrosine in Human Urine for Clinical Applications
Published on: July 14, 2017
T Brunelli1, G Pepe, R Marcucci
1Dipartimento di Area Critica Medico-Chirugica, Università di Firenze, Italy. labctrombosi@ao-careggi.toscana.it
Insights
Hyperhomocysteinemia increases atherothrombotic disease risk. The fluorescence polarization immunoassay (FPIA) method is suitable for clinical laboratories to measure homocysteine plasma levels, showing good agreement with the reference method.
Area of Science:
- Clinical Chemistry
- Biomarker Analysis
- Cardiovascular Disease Risk Assessment
Background:
- Hyperhomocysteinemia is a recognized risk factor for atherothrombotic diseases.
- Increasing demand for homocysteine testing necessitates reliable and accessible laboratory methods.
- Accurate measurement of homocysteine is crucial for assessing individual cardiovascular risk profiles.
Purpose of the Study:
- To compare the performance of two commercial immunoassays (EIA and FPIA) against a reference HPLC method for homocysteine plasma level determination.
- To evaluate the suitability of the FPIA assay for routine use in clinical laboratories.
Main Methods:
- Analysis of 100 plasma samples using an in-house high-pressure liquid chromatography (HPLC) method as the reference.
- Measurement of homocysteine levels using a commercial enzyme-linked immunoassay (EIA) and an automated fluorescence polarization immunoassay (FPIA).
- Statistical analysis including correlation and Bland-Altman analysis to assess agreement between methods.
Main Results:
- FPIA demonstrated the lowest within-run (3.6%) and between-run (4.1%) coefficients of variation.
- Significant correlations were observed between EIA and HPLC (r=0.81) and between FPIA and HPLC (r=0.85).
- Bland-Altman analysis indicated that FPIA showed the best agreement with the HPLC reference method, while EIA had a wider scatter.
Conclusions:
- The fluorescence polarization immunoassay (FPIA) exhibits excellent precision and strong correlation with the HPLC reference method.
- The technological characteristics of the FPIA assay make it a suitable and reliable method for homocysteine determination in clinical laboratory settings.
- FPIA offers a viable option for laboratories needing to accurately assess homocysteine levels for cardiovascular risk evaluation.
Abstract:
Hyperhomocysteinemia is a well established risk factor for atherothrombotic disease, and the request for homocysteine determinations and the number of laboratories that need to perform this assay to assess individual risk profile is increasing. Different methods to evaluate homocysteine plasma levels are at present available. In the present study three methods, an in-house high-pressure liquid chromatographic (HPLC) method (considered as reference method) and two commercial immunoassays, an enzyme-linked immunoassay (EIA) and an automated fluorescence polarization immunoassay (FPIA), were used to measure homocysteine plasma levels in 100 samples. The median of homocysteine plasma levels obtained by HPLC was 9.0 micromol/L (range 4.2-23.0); the median of values obtained by EIA and FPIA were 10.6 micromol/L (range 3.3-21.5) and 9.6 micromol/L (4.8-20.2), respectively. The FPIA method showed the lowest within-run and between-run coefficients of variation (3.6% and 4.1%, respectively). There was a significant correlation between EIA and HPLC (r=0.81; p<0.0001), and between FPIA and HPLC (r=0.85; p<0.0001). The Bland-Altman analysis showed that FPIA agreed best with HPLC; EIA displayed a relatively wide scatter of difference data points. The present results indicate that the technological characteristics of the FPIA assay make this method suitable for the determination of Hcy in clinical laboratories.
More Related Videos
05:08Measurement of Tissue Non-Heme Iron Content using a Bathophenanthroline-Based Colorimetric Assay
Published on: January 31, 2022
10:38Simultaneous Quantification of Selected Kynurenines Analyzed by Liquid Chromatography-Mass Spectrometry in Medium Collected from Cancer Cell Cultures
Published on: May 9, 2020