| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
|
|
|
|||||||
|
|||||||||
Letters to the Editor |
1 UMR INSERM unit 557, INRA unit 1125. Institut Scientifique et Technique, de la Nutrition et de l’Alimentation, ISTNA/CNAM, Paris, 75003 France
2 Département de Biologie Intégrée, CHU, Grenoble, 38043 France
3 Unité de Surveillance, et d’Epidémiologie Nutritionnelle, InVS-CNAM, Paris, 75003 France
4 Département de Médicine Interne, CHU Saint Eloi, Montpellier, 34059 France
aAddress correspondence to this author at: ISTNA/CNAM, 5 rue du Vertbois, 75003 Paris, France. Fax 33-1-53018070; e-mail s_debree{at}vcnam.cnam.fr.
To the Editor:
Increased plasma total homocysteine (tHcy) is a recognized risk factor for cardiovascular diseases (1)(2)(3). Recommendations for accurate measurement of tHcy concentrations include collecting fasting blood samples (4)(5) with the patient in a sitting position (6), placing the (EDTA) tubes directly on ice, and immediately centrifuging the samples (4). There are no recommendations on whether to use a refrigerated or a nonrefrigerated centrifuge. We compared the tHcy concentrations in samples centrifuged at 4 °C and at ambient temperature.
We studied 40 females (age range, 43–68 years) who participated in a side protocol of the SU.VI.MAX study (7). Two fasting venous blood samples were collected in EDTA Vacutainer Tubes (Becton Dickinson), which were not put on ice, but were centrifuged immediately for 15 min at 2000g, one at 4 °C and the other at ambient temperature (
20 °C). After centrifugation the plasma was immediately separated from the blood cells, and the plasma samples were stored at -20 °C until the tHcy measurement.
The tHcy concentration, including the protein-bound and non-protein-bound fractions of homocysteine, was measured by HPLC with fluorescence detection (8). The within- and between-run CVs for this method were 3.4% and 4.8%, respectively, at a tHcy concentration of 8.4 µmol/L (5).
Stability studies indicate that the increase in tHcy in whole blood kept at room temperature (20–25 °C) may be 0.009–0.023 µmol/L per min (9)(10)(11)(13). This means that if there is an effect of centrifugation at 20 °C, the expected increase in tHcy concentration in the samples centrifuged at room temperature would be 0.14–0.35 µmol/L because we centrifuge for 15 min. With 40 women the study would be able to detect an increase of 0.20 µmol/L with a statistical power of 80% and a one-sided 
of 0.10, with an assumed SD of the difference in tHcy concentrations of the matched pairs of 0.5 µmol/L.
A priori, a difference in the tHcy concentration <0.2 µmol/L was not regarded as relevant regarding misclassification of an individual with respect to risk of ischemic heart disease or stroke. The relevance of the difference was calculated with the data from a recent metaanalysis (2). From these data we can deduce that an increase in the tHcy concentration of 0.2 µmol/L is associated with an increase in risk of only 1% for ischemic heart disease and for stroke. A difference in the tHcy concentration <0.2 µmol/L would lead to a negligible increase in the risk of cardiovascular diseases; it was therefore not considered relevant.
All statistical analyses were done with the statistical package Analyze-it for Microsoft Excel (97 SR-2). Our results were based on data of 39 women (plasma was absent in one tube). The mean (SD) tHcy concentrations were 9.18 (3.7) µmol/L for the samples centrifuged at 4 °C and 9.25 (4.1) µmol/L for the samples centrifuged at 20 °C. The tHcy concentration of the samples centrifuged at 20 °C was thus on average 0.07 µmol/L higher. However, this somewhat higher value was not statistically significantly different from zero (95% confidence interval, -0.201 to 0.340), indicating no difference in tHcy concentration according to the centrifugation temperature. These results are supported by the Bland–Altman plot in Fig. 1
, which shows the difference between the tHcy concentrations in samples centrifuged at 4 and 20 °C for each woman plotted against the mean tHcy concentration at the two different temperatures.
|
We conclude that use of nonrefrigerated centrifuges does not lead to misclassification of individuals according to their tHcy concentration if whole blood is centrifuged immediately after drawing. If immediate centrifugation is not feasible, whole blood samples should be placed on ice immediately after drawing.
Acknowledgments
This work was supported financially by a grant from the Ministry of Research NUTRIALIS (réseau RARE, no. 02 P 0565).
References
T polymorphism and risk of coronary heart disease: a meta-analysis. JAMA 2002;288:2023-2031.
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
