BACKGROUND: It is well known that elevated level of total serum cholesterol represents a risk factor to developing cardiovascular disease (CVD). The present study aims to quantify total cholesterol in dried blood spot (DBS) by direct enzymatic method. METHODS: We selected the samples using serum cholesterol concentrations analyzed by automatic enzymatic colorimetric method (CHOD/POD/TRINDER, Roche Modular) (reference method). We kept 317 whole blood tubes of patients with serum cholesterol levels between 81 and 337 mg/dL, blood spots were prepared by 70 μL spotted on paper. The cholesterol was extracted from DBS using methanol and its concentration measured by manual enzymatic colorimetric method (CHOD/POD/TRINDER Roche). The extraction recovery of cholesterol from DBS was evaluated by GC-FID analysis. Method imprecision was evaluated on two DBS pools at two concentration levels. DBS cholesterol concentrations were correlated with those measured on serum by reference method. The correlation curve obtained was used to predict serum cholesterol concentration from DBS values measured in samples of 58 healthy volunteers. RESULTS: Imprecision study showed a within-day CV% of 3.98 (n=5; low level) and 7.69 (n=5; high level) and a between- day CV% of 5.97 (n=30; low level) and 6.32 (n=30; high level). Accuracy study showed a bias% of -12. The residual cholesterol on DBS disk after extraction was about 16%. The correlation study between reference method (on serum) and manual enzymatic method (on DBS) was linear (y= 0.4963x + 70.706; r = 0.9139). DBS cholesterol concentrations from 58 healthy volunteers were transformed using the correlation study to obtain serum cholesterol values, after, they were correlated with the corresponding plasma levels measured by reference method. The differences between two data groups were not statistically significant (p = 0.9487, Student’s T-test), and the Passing & Bablock regression comparison showed a high correlation (slope 1.0656, 95% CI 0.7931 to 1.4063, intercept –17.0608, 95% CI –77.7188 to 32.2586) and a bias % of -0.16. CONCLUSIONS: The obtained results show that this procedure is suitable to analyze DBS cholesterol and can be automated for populations screening.

Direct enzymatic analysis of cholesterol in dried blood spot

DELLO RUSSO, ANTONIO;GALLO, MONICA;GELZO, MONICA;GRAF, MARIA;SCARPATO, NICOLA;
2014

Abstract

BACKGROUND: It is well known that elevated level of total serum cholesterol represents a risk factor to developing cardiovascular disease (CVD). The present study aims to quantify total cholesterol in dried blood spot (DBS) by direct enzymatic method. METHODS: We selected the samples using serum cholesterol concentrations analyzed by automatic enzymatic colorimetric method (CHOD/POD/TRINDER, Roche Modular) (reference method). We kept 317 whole blood tubes of patients with serum cholesterol levels between 81 and 337 mg/dL, blood spots were prepared by 70 μL spotted on paper. The cholesterol was extracted from DBS using methanol and its concentration measured by manual enzymatic colorimetric method (CHOD/POD/TRINDER Roche). The extraction recovery of cholesterol from DBS was evaluated by GC-FID analysis. Method imprecision was evaluated on two DBS pools at two concentration levels. DBS cholesterol concentrations were correlated with those measured on serum by reference method. The correlation curve obtained was used to predict serum cholesterol concentration from DBS values measured in samples of 58 healthy volunteers. RESULTS: Imprecision study showed a within-day CV% of 3.98 (n=5; low level) and 7.69 (n=5; high level) and a between- day CV% of 5.97 (n=30; low level) and 6.32 (n=30; high level). Accuracy study showed a bias% of -12. The residual cholesterol on DBS disk after extraction was about 16%. The correlation study between reference method (on serum) and manual enzymatic method (on DBS) was linear (y= 0.4963x + 70.706; r = 0.9139). DBS cholesterol concentrations from 58 healthy volunteers were transformed using the correlation study to obtain serum cholesterol values, after, they were correlated with the corresponding plasma levels measured by reference method. The differences between two data groups were not statistically significant (p = 0.9487, Student’s T-test), and the Passing & Bablock regression comparison showed a high correlation (slope 1.0656, 95% CI 0.7931 to 1.4063, intercept –17.0608, 95% CI –77.7188 to 32.2586) and a bias % of -0.16. CONCLUSIONS: The obtained results show that this procedure is suitable to analyze DBS cholesterol and can be automated for populations screening.
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Utilizza questo identificativo per citare o creare un link a questo documento: https://hdl.handle.net/11588/588222
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