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Effects of anticoagulant and time of plasma separation on measurement of homocysteine.

Current interest in plasma total homocysteine (tHcy) measurements has increased with evidence that even mild hyperhomocysteinemia may be an independent risk factor for vascular disease (1) and that increases occur with vitamin deficiencies and reduced renal function. This has led to the investigation of problems associated with measurement, in particular the stability of tHcy after blood collection (2,3). Transfer of homocysteine from red cells to plasma after venesection may occur and produce a 10% per hour increase in plasma tHcy concentrations (4), and thus influence assessments of the relative risk of disease (5). To reduce this increase, samples may be stored on ice and centrifuged within 1 h of collection. In epidemiologic surveys or even routine collection, this may not always be feasible.

We studied the stability of plasma tHcy measured by a fluorescence polarization immunoassay (FPIA) in samples from 9 cystathionine [beta]-synthase (C[beta]S)-deficient homocystinuric patients and 13 healthy individuals. Blood was collected into both EDTA (1.8 g/L) and NaF (1.8 g/L EDTA and 3 g/L NaF) tubes. For six of the healthy individuals, we also used lithium heparin (14 kIU/L), sodium citrate (32 g/L trisodium citrate + 4.2 g/L citric acid; 3.2% solution), and ACD-B (13.2 g/L trisodium citrate + 5.25 g/L citric acid + 14.7 g/L glucose; 1.3% solution) tubes. The C[beta]S-deficient patients were diagnosed and treated as described previously (6, 7), and the controls were healthy staff. Informed consent was obtained from all participants.

Within 15 min of collection, samples were divided into five aliquots. One aliquot from each sample was centrifuged (10 000g for 5 min) 15 min after blood collection, and the plasma was separated. Aliquots from the remaining samples were incubated at room temperature (25 [+ or -] 3[degrees]C, mean [+ or -] SD) and centrifuged after specified lengths of time. Plasma was stored at -70[degrees]C before analysis with the Abbott (Abbott Diagnostics) IMx analyzer (8). Our interassay CVs were 5.7%, 4.7%, and 4.6% at 7.0, 12.5, and 25.0 [micro]mol/L tHcy, respectively.

tHcy concentrations at different time points were compared by repeated-measures ANOVA. The overall effects of anticoagulants on concentrations were assessed in the same model as a between-sample factor, and Bonferroni-corrected P values were reported. The differences in tHcy concentrations between time points were compared by the Student t-test. Percentage differences were calculated by comparison with baseline concentrations.


For the controls, baseline concentrations of plasma tHcy were significantly lower in NaF tubes than in EDTA tubes (P <0.001; Table 1). In EDTA tubes, tHcy increased significantly at 1 and 4 h, by 9% [+ or -] 4% and 27% [+ or -] 9%, respectively (P <0.001), for the controls, but did not increase significantly in the patients. Similar changes were seen with NaF tubes (Table 1).

A general linear model of repeated-measures ANOVA was applied to data for both controls and patients to evaluate the effects of storage on tHcy concentrations. The repeated measurements of tHcy at five different time points were the within-subject factors. In the controls, the within-subject factor, i.e., the duration of storage, significantly affected tHcy concentrations (F = 77.489; n = 13; P = 0.0001). For patients, the duration of storage was not significantly predictive of the tHcy concentrations (F = 0.783; P = 0.555).

With the same analytical model, we evaluated the effects of anticoagulants on tHcy during storage. Baseline concentrations in the six control individuals for the samples in EDTA, lithium heparin, sodium citrate, and ACD-B tubes were 8.1 [+ or -] 2.6, 7.9 [+ or -] 2.5, 7.7 [+ or -] 2.6, and 7.3 [+ or -] 2.6 [micro]mol/L, respectively (EDTA vs ACD-B, P = 0.014). There was no interaction between the duration of storage and anticoagulant used for sample collection (F = 1.296; P = 0.221). After Bonferroni correction, the differences in tHcy concentrations among different anticoagulants were not statistically significant (P >0.05; n = 6 for all comparisons), and there was also no interactive effect between duration of storage and anticoagulants (F = 0.566; P = 0.858). After Bonferroni correction, the effects of anticoagulants on measured tHcy concentrations were not statistically significant. Percentage increases over the next 4 h were similar for each: at 1 h, 8% [+ or -] 2%; at 2 h, 12% [+ or -] 2%; at 3 h, 18% [+ or -] 2%; and 4 h, 24% [+ or -] 2%. Fig. 1A shows the tHcy concentrations with each anticoagulant over 4 h in each individual. tHcy measurements were corrected for the ratio of anticoagulant to whole blood (1:4 for the ACD-B and 1:10 for the citrate collection tube) (9). (EDTA in the crystalline form in tubes is assumed to produce no dilution, ignoring water shifts between cells and plasma.) There was a uniform pattern of increased concentrations with delayed separation in the samples from controls.

The tHcy values in the patient samples collected into EDTA and NaF over 4 h from separation are shown in Fig. 1B. Concentrations were consistently lower in samples collected into NaF tubes. With both collection methods, tHcy concentrations ranging from 25 to 120 [micro]mol/L were unaffected by the delay in separation.

When tHcy concentrations are within the reference interval, our results are consistent with a release of homocysteine in the tested types of collection tubes. These findings differ from those of Palmer-Toy et al. (9 ) and Willems et al. (10 ), who found that tHcy concentrations were stable in blood collected into sodium citrate tubes and maintained at room temperature for 6 h. They also differ from the observations of Moller and Rasmussen (11), who reported that collection into NaF tubes hinders homocysteine release for 2 h at room temperature. However, they are consistent with the findings of Caliskan et al. (12) and Salazar et al. (2).

In the C[beta]S-deficient patients, we found no significant increase over 4 h in measured plasma tHcy with concentrations [greater than or equal to] 25.0 [micro]mol/L. Our findings suggest that at these concentrations plasma and circulating blood cell concentrations are in equilibrium, but not when tHcy is within the reference interval.

Plasma collected into NaF tubes had tHcy concentrations 16.6% [+ or -] 1.6% lower than in the EDTA tubes at baseline, and the increases in concentrations with time in healthy individuals for NaF tubes were ~2% lower than for the EDTA tubes. This may be attributable to a slower rate of tHcy release from red blood cells, perhaps by inhibition of glycolysis, or to some interaction with the assay.

We conclude that with the Abbott method, measured tHcy concentrations within the reference interval are lower for blood collected into NaF (P <0.001) and ACD-B tubes than for blood collected into EDTA, lithium heparin, and sodium citrate tubes. Although delayed separation increases tHcy when concentrations are within the reference interval, markedly increased tHcy concentrations ([greater than or equal to] 25 [micro]mol/L) remain stable at room temperature for up to 4 h, suggesting that concentrations within red cells and plasma are then in equilibrium and that further tHcy production in red cells is not occurring. It is also possible that the release of tHcy from red blood cells is independent of plasma tHcy concentrations. When a fixed amount of tHcy is released into plasma, the concentration changes produced by this addition could be analytically significant when the base plasma concentrations are low, as in the healthy controls, but have minimal effects when base concentrations are high, as in the patients. Thus, reference values for tHcy need to be defined in relation to the method of sample collection, the time to separation, and the assay system used.


(1.) Welch GN, Loscalzo J. Homocysteine and atherothrombosis. N Engl J Med 1998;338:1042-50.

(2.) Salazar JF, Herbeth B, Siest G, Leroy P. Stability of blood homocysteine and other thiols: EDTA or acidic citrate? Clin Chem 1999;45:2016-9.

(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.) Andersson A, Isaksson A, Hultberg B. Homocysteine export from erythrocytes and its implication for plasma sampling. Clin Chem 1992;38:1311-5.

(5.) Eliason SC, Ritter D, Chung HD, Creer M. Interlaboratory variability for total homocysteine analysis in plasma [Letter]. Clin Chem 1999;45:315-6.

(6.) Wilcken DE, Wilcken B. The natural history of vascular disease in homocystinuria and the effects of treatment. J Inherit Metab Dis 1997;20:295-300.

(7.) Wilcken DE, Wang XL, Adachi T, Hara H, Duarte N, Green K, et al. Relationship between homocysteine and superoxide dismutase in homocystinuria: possible relevance to cardiovascular risk. Arterioscler Thromb Vasc Biol 2000;20:1199-202.

(8.) Nexo E, Engbaek F, Ueland PM, Westby C, O'Gorman P, Johnston C, et al. Evaluation of novel assays in clinical chemistry: quantification of plasma total homocysteine. Clin Chem 2000;46:1150-6.

(9.) Palmer-Toy DE, Szczepiorkowski ZM, Shih V, Van Cott EM. Compatibility of the Abbott IMx homocysteine assay with citrate-anticoagulated plasma and stability of homocysteine in citrated whole blood. Clin Chem 2001;47: 1704-7.

(10.) Willems HP, Bos GM, Gerrits WB, den Heijer M, Vloet S, Blom HJ. Acidic citrate stabilizes blood samples for assay of total homocysteine. Clin Chem 1998;44:342-5.

(11.) Moller J, Rasmussen K. Homocysteine in plasma: stabilization of blood samples with fluoride. Clin Chem 1995;41:758-9.

(12.) Caliskan S, Kuralay F, Onvural B. Effect of anticoagulants on plasma homocysteine determination. Clin Chim Acta 2001;309:53-6.

Natalia Louise Duarte, [1] Xing Li Wang, [2] and David Emil Leon Wilcken [1] * ([1] Cardiovascular Genetics Laboratory, Department of Medicine, Prince of Wales Hospital, Randwick, NSW 2031 Australia; [2] Department of Genetics, Southwest Foundation for Biomedical Research, San Antonio, TX 78245- 0549; * author for correspondence: fax 612-9382-4921, e-mail
Table 1. Changes of plasma tHcy ([micro]mol/L) in 9
C[beta]S-deficient patients and 13 healthy controls over time
from venesection to separation in blood collected into EDTA
and NaF tubes.

 Time to separation, h

 0 1

Patients (a)
 EDTA 90 [+ or -] 32 91 [+ or -] 31
 2% (4%)
 NaF 79 [+ or -] 28 79 [+ or -] 29
 0% (8%)

Controls (a)
 EDTA 9.7 [+ or -] 2.6 10.6 [+ or -] 2.8
 9% (4%) (b)
 NaF 8.3 [+ or -] 2.2 8.9 [+ or -] 2.2
 8% (6%) (b)

 Time to separation, h

 2 3

Patients (a)
 EDTA 88 [+ or -] 31 91 [+ or -] 31
 0% (7%) 2% (4%)
 NaF 77 [+ or -] 29 84 [+ or -] 34
 -2% (8%) 6% (12%)

Controls (a)
 EDTA 11.2 [+ or -] 3.0 11.8 [+ or -] 3.2
 16% (6%) (b) 22% (8%) (b)
 NaF 9.1 [+ or -] 2.1 9.6 [+ or -] 2.2
 11% (7%) (b) 17% (10%) (b)

 Time to
 separation, h


Patients (a)
 EDTA 94 [+ or -] 33
 5% (7%)
 NaF 80 [+ or -] 30
 1% (7%)

Controls (a)
 EDTA 12.3 [+ or -] 3.2
 27% (9%) (b)
 NaF 10.2 [+ or -] 2.7
 24% (13%) (b)

(a) Values are mean [+ or -] SD, with percentage change (SD)
shown below each value.

(b) P <0.001.
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Title Annotation:Technical Briefs
Author:Duarte, Natalia Louise; Wang, Xing Li; Wilcken, David Emil Leon
Publication:Clinical Chemistry
Date:Apr 1, 2002
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