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Letters to the Editor |
1 Department of Obstetrics and Gynaecology, University of Geneva, Geneva, Switzerland
aAddress correspondence to this author at: Laboratoire d’Hormonologie, Maternité, 1211 Geneva 14, Switzerland. Fax 41-22-382-4310; e-mail paul.bischof{at}hcuge.ch.
To the Editor:
Matrix metalloproteinases (MMPs) are a family of zinc endopeptidases collectively capable of degrading essentially all components of the extracellular matrix. They are involved in many physiologic and pathologic processes, such as wound healing, angiogenesis, embryo implantation, cancer progression, and metastasis. Several studies have measured circulating MMP-2 and -9 in cancer patients, but the results have been contradictory, specially for MMP-9, for which very large patient-to-patient variability was observed (1)(2). This could be caused by different preanalytical conditions in blood sampling, as suggested by recent studies (3)(4)(5)(6). We studied the impact of blood sampling conditions on the measurement by ELISA of MMP-2 and -9. This study was accepted by our Institutional Review Board, and written informed consent was obtained from all volunteers participating in the study.
In our first study, we collected venous blood samples from 12 healthy volunteers into VacutainerTM Tubes containing clot activator (SST), lithium heparinate (LH), dipotassium EDTA, or sodium citrate. The tubes were either centrifuged immediately (t0) or after 0.5, 2, or 24 h. Tubes were left at room temperature, except for the tubes that sat for 24 h, which were kept at 4 °C. Measurements were performed within 5 days of sampling, and plasma and sera were stored at 4 °C until assayed.
In our second study, we collected venous blood samples from four healthy volunteers as described above (except for serum). Cells and plasma were immediately separated by centrifugation and aspiration. Cells were then transferred to clean plastic tubes containing a volume of sterile saline equal to that of the original plasma. Plasma and cells were incubated for 0.5 h, 2 h (room temperature) and 24 h (4 °C). After incubation, plasma and cell supernatants were stored at –20 °C until assayed.
MMP-2 and -9 concentrations were measured in plasma and serum samples by our own enzyme immunoassays (EIAs) (7). Results were confirmed by commercially available assays (Biotrak; Amersham). Gelatinolytic activity was measured by zymography as described previously (8).
Statistical analyses were performed by ANOVA using the Fisher test. P <0.05 was considered as representing a statistically significant difference.
In the first study, with the exception of citrate plasma, the concentration of immunoreactive MMP-9 increased with the time between sampling and centrifugation (Fig. 1A
). The effect of time was dependent on the type of anticoagulant, however, being much more marked in heparin plasma and serum compared with EDTA plasma. Furthermore, the mean (SE) measured concentration of MMP-9 in EDTA plasma [55.4 (8.1 µg/L)] was significantly higher than in citrate plasma [19.4 (3.5) µg/L; P = 0.0003], heparin plasma [27.4 (3.1) µg/L; P = 0.0002], or serum [32.6 (1.2) µg/L; P = 0.0094].
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We obtained similar results when we measured the gelatinolytic activity of MMP-9 by zymography (results not shown), except that MMP-9 activity was significantly increased after 2 h only in heparin plasma and serum.
In sharp contrast to MMP-9, the concentration of immunoreactive MMP-2 did not increase with time between sampling and centrifugation; in fact, it was decreased in EDTA plasma after 2 and 24 h. The measured concentrations of MMP-2 were significantly lower in EDTA plasma [86.5 (15.6) µg/L] than in citrate plasma [405.5 (54.3) µg/L; P <0.0001], heparin plasma [569.9(26.5) µg/L; P <0.0001], and serum [610.6 (25.9) µg/L; P <0.0001].
Addition of increasing concentration of EDTA (final concentration, 0.06–1.8 g/L) to serum markedly reduced the measured concentration of MMP-2, whereas it had no effect on MMP-9 (results not shown).
In the second study, the concentration of MMP-9 released by blood cells separated immediately from citrate, EDTA, and heparin plasma increased with the time of incubation of the cells in saline (Fig. 1B
). In contrast, MMP-9 concentrations in the plasmas (from which the cells were isolated) incubated for the same period of time did not change over time (results not shown). MMP-2 concentrations also did not change over time in the supernatant of incubated cells (Fig. 1B
) or in plasma (results not shown).
Preanalytical conditions affect circulating MMP-2 and -9 concentrations. Jung et al. (4) have shown, by EIA, that MMP-9 was higher in serum than in heparin plasma and that MMP-2 was lowered in the presence of EDTA (3). Mannello et al. (5) showed by zymography that MMP-9 was higher in serum than in heparin or citrate plasma and that addition of EDTA decreased the concentrations of MMP-2 and increased those of MMP-9. Finally, Makowski et al. (6) showed by zymography that MMP-9 was higher in heparin and EDTA plasma than in citrate plasma when blood was left 1 h between sampling and centrifugation.
Using a quantitative assay (EIA) to measure the MMP concentrations, we confirmed the differences between the anticoagulants and the importance of controlling time between blood sampling and centrifugation. We showed that these differences are attributable to the release of MMPs by blood cells. In serum and heparin plasma, the release of MMP-9 is massive and time-dependent, rendering any clinical study impossible, whereas MMP-2 is not affected. EDTA markedly decreases the concentration of MMP-2, whereas it increases MMP-9. We conclude that citrate is the anticoagulant of choice because it inhibits the release of gelatinases by blood cells in the plasma and thus reduces the influence of time.
References
, interleukin-1 , macrophage colony stimulating factor and transforming growth factor ß on trophoblastic matrix metalloproteinases. Mol Hum Reprod 1999;5:252-260.The following articles in journals at HighWire Press have cited this article:
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  K. Jung Consideration of preanalytical impact of blood sampling on measurement of matrix metalloproteinases and their inhibitors as precondition to evaluate their relationship to clinical data Multiple Sclerosis, November 1, 2009; 15(11): 1372 - 1373. [PDF]
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Y. Benesova Matrix metalloproteinase-9 and matrix metalloproteinase-2 as biomarkers of various courses in multiple sclerosis Multiple Sclerosis, November 1, 2009; 15(11): 1374 - 1375. [PDF]
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 A. Laverriere, R. Landau, I. Charvet, O. Irion, P. Bischof, M. Morales, and M. Cohen GRP78 as a marker of pre-eclampsia: an exploratory study Mol. Hum. Reprod., September 1, 2009; 15(9): 569 - 574. [Abstract] [Full Text] [PDF]
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 F. Mannello Serum or Plasma Samples?: The "Cinderella" Role of Blood Collection Procedures Preanalytical Methodological Issues Influence the Release and Activity of Circulating Matrix Metalloproteinases and Their Tissue Inhibitors, Hampering Diagnostic Trueness and Leading to Misinterpretation Arterioscler Thromb Vasc Biol, April 1, 2008; 28(4): 611 - 614. [Full Text] [PDF]
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K. Jung Matrix Metalloproteinase-8 and Tissue Inhibitor of Metalloproteinase-1 in Serum Do Not Reflect the Analytes Circulating in Blood Arterioscler Thromb Vasc Biol, March 1, 2008; 28(3): e15 - e16. [Full Text] [PDF]
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F. Mannello, G.A.M. Tonti, and F. Canestrari The `never-ending story' of the influence of blood specimen collection methods affecting the concentration, the zymographic profile and the usefulness of matrix metalloproteinases and their tissue inhibitors in multiple sclerosis diagnosis/prognosis: a landmark for limiting the misuse of serum samples Multiple Sclerosis, June 1, 2007; 13(5): 687 - 690. [PDF]
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 K. Jung Impact of Blood Sampling on Circulating Tissue Inhibitors of Metalloproteinases Clin. Cancer Res., April 15, 2006; 12(8): 2648 - 2648. [Full Text] [PDF]
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