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Clinical Chemistry 50: 1881-1883, 2004; 10.1373/clinchem.2004.040634
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(Clinical Chemistry. 2004;50:1881-1883.)
© 2004 American Association for Clinical Chemistry, Inc.

Letters to the Editor

Measurement of Total Homocysteine Concentrations in Acidic Citrate- and EDTA-Containing Tubes by Different Methods

Huub P.J. Willems1,a, Martin den Heijer2, Jan Lindemans3, Henriette W.A. Berenschot4, Wim B.J. Gerrits1, Gerard M.J. Bos5 and Henk J. Blom6

1 Department of Hematology, Leyenburg Hospital, The Hague, The Netherlands
2 Departments of Endocrinology and Epidemiology, and Biostatistics and6 Laboratory of Pediatrics and Neurology, University Medical Center Nijmegen, Nijmegen, The Netherlands
3 Laboratory of Clinical Chemistry, Erasmus Medical Center, Rotterdam, The Netherlands
4 Department of Internal Medicine, Albert Schweitzer Hospital, Dordrecht, The Netherlands
5 Department of Internal Medicine, University Hospital Maastricht, Maastricht, The Netherlands

aAddress correspondence to this author at: Department of Internal Medicine, Máxima Medical Center Eindhoven, PO Box 90052, 5600 PD Eindhoven, The Netherlands. Fax 31-40-245-0385; e-mail hpj.willems{at}niv.medonline.nl.


To the Editor:

Blood collection for total homocysteine (tHcy) measurements is usually done in tubes containing EDTA as anticoagulant. At room temperature there is an increase in tHcy in whole blood (1)(2); therefore, tubes must be put on melting ice immediately and centrifuged within 1 or 2 h. Large epidemiologic studies, however, require blood collection techniques that are easily applicable in a field setting. We found that citrate with a low pH (pH 4.3; pH ~5.9 after mixing with blood) stabilizes tHcy in whole blood for 6 h when the blood is stored at room temperature (1), but that measured absolute tHcy concentrations differ in acidic citrate and EDTA.

In this study we investigated the differences between measured tHcy concentrations in EDTA- and acidic citrate-anticoagulated blood obtained with different measurement methods. Blood was collected from 208 volunteers (79 males and 129 females; age range, 23–88 years; median age, 65 years) in tubes containing EDTA (Vacutainer®; Becton Dickinson) and tubes containing acidic citrate (Stabilyte®; Biopool) as anticoagulant. The EDTA tubes were stored at 0 °C immediately after blood sampling until processing, whereas the acidic citrate tubes were kept at room temperature. tHcy was measured with three different methods:

As shown in Table 1 , there were differences between mean tHcy concentrations measured in acidic citrate and EDTA plasma, but tHcy in acidic citrate and EDTA correlated highly regardless of the measurement method used. We hypothesized that the differences found could be the result of the acidic environment interacting with the measurement. We acidified EDTA plasma by adding HCl and did 10 additional measurements with the HPLC(a) method, but measured tHcy concentrations in the regular EDTA plasma and the acidic EDTA plasma were comparable (data not shown). It may be that acidic citrate itself either inhibits derivatization with ammonium-7-fluorobenzo-2-oxa-1,3-diazole-4-sulfonate, causing the tHcy to be lower with the HPLC(b) method, or causes some interference with the HPLC(a) method, leading to higher measured concentrations in the acidic citrate tubes. This idea is supported by the fact that ammonium-7-fluorobenzo-2-oxa-1,3-diazole-4-sulfonate gives rather "clean" chromatograms, whereas monobromobimane contains spurious material that could generate possible interference.


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  		Table 1. Comparison of tHcy results in tubes containing acidic citrate and EDTA as anticoagulant.

The observed matrix effect is of utmost importance for external calibration of assays for tHcy measurement. Our data highlight the fact that a fluorescent assay for thiols based on derivatization cannot be used with different matrices without validation, which includes recovery experiments and tests for analytical interference.

On the basis of our results, we conclude that tubes containing acidic citrate can be used for homocysteine determination, especially in epidemi-ologic studies, because tHcy corre-lates highly with tHcy in EDTA plasma. However, reference values need to be established for each labo-ratory when either acidic citrate or EDTA is used as anticoagulant.


References

  1. 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;44:342-345.[Free Full Text]
  2. de Bree A, Verschuren WM, Blom HJ, Graaf-Hess A, Trijbels FJ, Kromhout D. The homocysteine distribution: (mis)judging the burden. J Clin Epidemiol 2001;54:462-469.[CrossRef][ISI][Medline] [Order article via Infotrieve]
  3. Fiskerstrand T, Refsum H, Kvalheim G, Ueland PM. Homocysteine and other thiols in plasma and urine: automated determination and sample stability. Clin Chem 1993;39:263-271.[Abstract]
  4. te Poele Pothoff MT, van den Berg M, Franken DG, Boers GH, Jakobs C, de Kroon IF, et al. Three different methods for the determination of total homocysteine in plasma. Ann Clin Biochem 1995;32:218-220.
  5. Araki A, Sako Y. Determination of free and total homocysteine in human plasma by high-performance liquid chromatography with fluorescence detection. J Chromatogr 1987;422:43-52.[ISI][Medline] [Order article via Infotrieve]
  6. Ubbink JB, Hayward Vermaak WJ, Bissbort S. Rapid high-performance liquid chromatographic assay for total homocysteine levels in human serum. J Chromatogr 1991;565:441-446.[ISI][Medline] [Order article via Infotrieve]
  7. Shipchandler MT, Moore EG. Rapid, fully automated measurement of plasma homocyst(e)ine with the Abbott IMx analyzer. Clin Chem 1995;41:991-994.[Abstract/Free Full Text]
  8. Bland JM, Altman DG. Statistical methods for assessing of agreement between two methods of clinical measurement. Lancet 1986;1:307-310.[CrossRef][ISI][Medline] [Order article via Infotrieve]



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Stability of Plasma Homocysteine, S-Adenosylmethionine, and S-Adenosylhomocysteine in EDTA, Acidic Citrate, and PrimavetteTM Collection Tubes
Clin. Chem., December 1, 2007; 53(12): 2217 - 2218.
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