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Effects of prolonged ambient storage of sodium fluoride/heparin specimens on plasma homocysteine.

To the Editor:

Increased plasma homocysteine is regarded as a risk factor for arterial and venous occlusive disease. Sample collection ideally requires use of cooled EDTA containers and immediate centrifugation of the blood (1). These conditions may be hard to maintain in a clinic and are not possible in the many medical practices that lack a centrifuge. We tested whether analyzing uncentrifuged EDTA or NaF/heparin plasma bears a greater risk of giving false-positive homocysteine results under these conditions.

Blood was drawn from 50 healthy volunteers (40 female and 10 male) into 1 EDTA and 4 NaF/heparin Vacutainers (Becton Dickinson), which were stored at room temperature. The Vacutainers were randomly assigned to groups--EDTA-15min, NaF/heparin-15min, NaF/heparin-24h, NaF/ heparin-48h, and NaF/heparin-144h--and centrifuged at 2700g for 7 min under cooling after the time stated in the group name. Homocysteine was analyzed by liquid chromatographytandem mass spectrometry, as described by Arndt et al. (2). Statistical analyses (box-plots and AMOVA) were done with the Analyze-it software (Analyze-it Software Ltd.), setting significance at P <0.05.


We found (a) a significantly greater mean homocysteine concentration in EDTA-15min plasma (11.5 [micro]mol/L) than in NaF/heparin-15 min plasma (9.5 [micro]mol/L) and (b) a significant increase in homocysteine in NaF/heparin-15-min (9.5 [micro]tmol/L) compared with NaF/heparin-24h (11.5 [micro]mol/L, which is equal to the EDTA-15min value) but no further increase with greater time gaps between blood sampling and centrifugation for NaF/heparin blood (Fig. 1).

Our data show higher homocysteine results in EDTA-15min plasma than in NaF/heparin-15min samples. Plasma dilution by NaF-induced water loss from the erythrocytes has been reported (1, 3). Hughes et al. (3) found lower mean homocysteine concentrations in NaF/heparin plasma stored at ambient temperature than in EDTA blood stored at 0[degrees]C; however, after -3 h, the difference in homocysteine concentrations was no longer significant (3). We conclude that NaF/heparin blood stored for at least 3 h at ambient temperature shows homocysteine concentrations comparable to those from EDTA blood centrifuged immediately after blood sampling. Dilution effects by NaF should no longer be significant after this time.

Our most important finding, which had not been shown previously, was that a prolonged time interval of 48 h (and 144 h) between blood sampling and centrifugation, which is common under routine conditions, is not accompanied by a further homocysteine increase in NaF/heparin plasma (Fig. 1). In contrast, EDTA blood shows a steady increase in plasma homocysteine reaching 380% after 168 h (4).

We conclude that in the absence of a centrifuge, NaF/heparin blood can be used for homocysteine analysis without the need for specific reference values. Therefore, we consider NaF/heparin blood to be a suitable material for homocysteine measurements in hospitals with commonly delayed sample transport to the in-house or reference laboratory or at medical practices without a centrifuge.


(1.) Refsum H, Smith AD, Ueland PM, Nexo E, Clarke R, McPartlin J, et al. Facts and recommendations about total homocysteine determinations: an expert opinion. Clin Chem 2004;50:3-32.

(2.) Arndt T, Guessregen B, Hohl A, Heicke B. Total plasma homocysteine measured by liquid chromatography-tandem mass spectrometry with use of 96-well plates. Clin Chem 2004;50: 755-7.

(3.) Hughes MP, Carlson TH, McLaughlin MK, Bankson DD. Addition of sodium fluoride to whole blood does not stabilize plasma homocysteine but produces dilution effects on plasma constituents and hematocrit. Clin Chem 1998;44: 2204-6.

(4.) Clark S, Youngman LID, Sullivan J, Peto R, Collins R. Stabilization of homocysteine in unseparated blood over several days: a solution for epidemiological studies. Clin Chem 2003;49:518-20.

DOI: 10.1373/clinchem.2005.052852

Ralf Scheidhauer * Brunhilde Guessregen Axel Hohl Torsten Arndt

Bioscientia GmbH Ingelheim, Germany

* Address correspondence to this author at: Bioscientia GmbH, Konrad-Adenauer-Strasse 17, D-55218 Ingelheim, Germany. Fax 49-6132-781-428; e-mail
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Title Annotation:Letters
Author:Scheidhauer, Ralf; Guessregen, Brunhilde; Hohl, Axel; Arndt, Torsten
Publication:Clinical Chemistry
Article Type:Letter to the editor
Date:Aug 1, 2005
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