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Serum or plasma: What kind of blood sample should be used to measure circulating matrix metalloproteinases and their inhibitors?
Journal of Neuroimmunology 162 (2005) 1 – 2 www.elsevier.com/locate/jneuroim
Letter to the Editor
Dear Sir,
0165-5728/$ - see front matter D 2005 Elsevier B.V. All rights reserved. doi:10.1016/j.jneuroim.2004.12.021
A: MMP-9 SERUM
750
b,c,d,e
100
PLASMA a,c,d,e
a,b,d,e
a,b,c,e
a,b,c,d
75
500
50
250
25
0
0 Serum-
3000
Serum+
Citrate
Heparin
EDTA
B: TIMP-1 SERUM d,e
600
PLASMA b
b
2000
400
1000
200
(µg/l)
Abbreviations: MMP-9, matrix metalloproteinase 9; TIMP-1, tissue inhibitor of metalloproteinase.
1000
(µg/l)
Recently, Blanco et al. (2004) reported on the concentrations of matrix metallproteinase 9 (MMP-9) and its inhibitor (TIMP-1) in serum of patients with multiple sclerosis. In comparison with controls, they found significantly increased serum levels of MMP-9 while the concentration of serum TIMP-1 was decreased and the ratio of MMP-9 to TIMP-1 was subsequently higher in patients than in controls. The authors discussed these changed serum concentrations as potential surrogate markers to characterize the activity of multiple sclerosis or the treatment effect. However, I suggest that Blanco et al. (2004) have rather inadequately taken into account the significance of blood collection as an important preanalytical determinant of MMP and TIMP results. Since there is rising evidence that blood sampling markedly determines the concentration of circulating MMP-9 I would draw the readers’ attention to these facts especially discussed in analytical journals (Jung et al., 1998; Jung et al., 1996; Mannello et al., 2003). A summarized report of own results of the effect of different blood sampling tubes on MMP-9 and TIMP-1 measurements is shown in Fig. 1. Briefly, venous blood samples from healthy volunteers (n=8 for MMP-9; n=10 for TIMP-1) were simultaneously collected in different devices for preparation of serum or plasma samples (Monovette tubes, Sarstedt, Nqrtingen, Germany). The tubes were centrifuged within 30 min after venipuncture at 1600g for 15 min at room temperature. MMP-9 and TIMP-1 were measured using commercially available ELISA kits (Medac Diagnostika, Wedel, Germany; Amersham, Little Chalfont, UK). MMP-9 concentrations in serum samples collected in tubes with clot activator were more than 3 fold higher than
(µg/l)
Keywords: MMP-9; TIMP-1; Blood sample; Preanalytical factors
in pure serum samples and essentially higher than the concentrations found in plasma samples (Fig. 1A). The TIMP-1 concentrations were also about 5–7 times higher in serum than in plasma (Fig. 1B). Since platelets and leukocytes contain high concentrations of MMP-9 and TIMP-1 (Kodama et al., 1990; Cooper et al., 1985), the varying release of these analytes blood cells during the platelet activation or sampling process could cause these differences (Mannello et al., 2003). In addition, changes of
(µg/l)
Serum or plasma: What kind of blood sample should be used to measure circulating matrix metalloproteinases and their inhibitors?
0
0
Serum+
Heparin
EDTA
Fig. 1. Effect of blood sampling on the MMP-9 and TIMP-1 concentration in serum and plasma. Median values and interquartile intervals are shown. MMP-9 and TIMP-1 were measured in samples prepared from blood of 8 and 10 healthy adults, respectively. Left (serum): serum , pure serum prepared in Monovette tubes without additive; serum+, serum prepared in tubes containing kaolin-coated granulate as clot activator. Right (plasma): citrate, heparin, and EDTA, plasma prepared in tubes coated with sodium citrate, lithium heparin, or K-EDTA. Significant differences of at least Pb0.05 (Wilcoxon rank test) between the samples are indicated by the following symbols: (a) from serum ; (b) from serum+; (c) from plasmacitrate; (d) from plasma-heparin; (e) from plasma-EDTA.
2
Letter to the Editor
white blood cell count are observed during the disease activity and the treatment of multiple sclerosis and could subsequently result in changed TIMP-1 concentrations when measurements are performed in serum. In summary, these important preanalytical conditions should be considered in the interpretation of changed MMP9 and TIMP-1 levels. Recently, the use of blood samples collected with sodium citrate was suggested to be the best procedure to avoid the detrimental effect of other anticoagulants or serum and to optimize the diagnostic validity of MMP-9 in peripheral blood (Mannello et al., 2003).
References Blanco, Y., Saiz, A., Carreras, E., Graus, F., 2004. Changes of matrix metalloproteinase-9 and its tissue inhibitor (TIMP-1) after autologous hematopoietic stem cell transplantation in multiple sclerosis. J. Neuroimmunol. 153, 190 – 194. Cooper, T.W., Eisen, A.Z., Stricklin, G.P., Welgus, H.G., 1985. Plateletderived collagenase inhibitor: characterization and subcellular localization. Proc. Natl. Acad. Sci. U. S. A. 82, 2779 – 2783. Jung, K., Nowak, L., Lein, M., Henke, W., Schnorr, D., Loening, S.A., 1996. What kind of specimen should be selected for determining tissue
inhibitor of metalloproteinase-1 (TIMP-1) in blood? Clin. Chim. Acta 254, 97 – 100. Jung, K., Laube, C., Lein, M., Lichtinghagen, R., Tschesche, H., Schnorr, D., Loening, S., 1998. Kind of sample as preanalytical determinant of matrix metalloproteinases 2 and 9 (MMP2; MMP9) and tissue inhibitor of metalloproteinase 2 (TIMP2) in blood. Clin. Chem. 44, 1060 – 1062. Kodama, S., Iwata, K., Iwata, H., Yamashita, K., Hayakawa, T., 1990. Rapid one-step sandwich enzyme immunoassay for tissue inhibitor of metalloproteinases. An application for rheumatoid arthritis serum and plasma. J. Immunol. Methods 127, 103 – 108. Mannello, F., Luchetti, F., Canonico, B., Papa, S., 2003. Effect of anticoagulants and cell separation media as preanalytical determinants on zymographic analysis of plasma matrix metalloproteinases. Clin. Chem. 49, 1956 – 1957.
Klaus Jung Department of Urology, University Hospital Charite´, Humboldt University Berlin, Schumannstrasse 20/21, D-10098 Berlin, Germany E-mail address: [email protected] Tel.: +49 30 450 515041/515127; fax: +49 30 450 515904. 9 December 2004
Letter to the Editor
Dear Sir,
0165-5728/$ - see front matter D 2005 Elsevier B.V. All rights reserved. doi:10.1016/j.jneuroim.2004.12.021
A: MMP-9 SERUM
750
b,c,d,e
100
PLASMA a,c,d,e
a,b,d,e
a,b,c,e
a,b,c,d
75
500
50
250
25
0
0 Serum-
3000
Serum+
Citrate
Heparin
EDTA
B: TIMP-1 SERUM d,e
600
PLASMA b
b
2000
400
1000
200
(µg/l)
Abbreviations: MMP-9, matrix metalloproteinase 9; TIMP-1, tissue inhibitor of metalloproteinase.
1000
(µg/l)
Recently, Blanco et al. (2004) reported on the concentrations of matrix metallproteinase 9 (MMP-9) and its inhibitor (TIMP-1) in serum of patients with multiple sclerosis. In comparison with controls, they found significantly increased serum levels of MMP-9 while the concentration of serum TIMP-1 was decreased and the ratio of MMP-9 to TIMP-1 was subsequently higher in patients than in controls. The authors discussed these changed serum concentrations as potential surrogate markers to characterize the activity of multiple sclerosis or the treatment effect. However, I suggest that Blanco et al. (2004) have rather inadequately taken into account the significance of blood collection as an important preanalytical determinant of MMP and TIMP results. Since there is rising evidence that blood sampling markedly determines the concentration of circulating MMP-9 I would draw the readers’ attention to these facts especially discussed in analytical journals (Jung et al., 1998; Jung et al., 1996; Mannello et al., 2003). A summarized report of own results of the effect of different blood sampling tubes on MMP-9 and TIMP-1 measurements is shown in Fig. 1. Briefly, venous blood samples from healthy volunteers (n=8 for MMP-9; n=10 for TIMP-1) were simultaneously collected in different devices for preparation of serum or plasma samples (Monovette tubes, Sarstedt, Nqrtingen, Germany). The tubes were centrifuged within 30 min after venipuncture at 1600g for 15 min at room temperature. MMP-9 and TIMP-1 were measured using commercially available ELISA kits (Medac Diagnostika, Wedel, Germany; Amersham, Little Chalfont, UK). MMP-9 concentrations in serum samples collected in tubes with clot activator were more than 3 fold higher than
(µg/l)
Keywords: MMP-9; TIMP-1; Blood sample; Preanalytical factors
in pure serum samples and essentially higher than the concentrations found in plasma samples (Fig. 1A). The TIMP-1 concentrations were also about 5–7 times higher in serum than in plasma (Fig. 1B). Since platelets and leukocytes contain high concentrations of MMP-9 and TIMP-1 (Kodama et al., 1990; Cooper et al., 1985), the varying release of these analytes blood cells during the platelet activation or sampling process could cause these differences (Mannello et al., 2003). In addition, changes of
(µg/l)
Serum or plasma: What kind of blood sample should be used to measure circulating matrix metalloproteinases and their inhibitors?
0
0
Serum+
Heparin
EDTA
Fig. 1. Effect of blood sampling on the MMP-9 and TIMP-1 concentration in serum and plasma. Median values and interquartile intervals are shown. MMP-9 and TIMP-1 were measured in samples prepared from blood of 8 and 10 healthy adults, respectively. Left (serum): serum , pure serum prepared in Monovette tubes without additive; serum+, serum prepared in tubes containing kaolin-coated granulate as clot activator. Right (plasma): citrate, heparin, and EDTA, plasma prepared in tubes coated with sodium citrate, lithium heparin, or K-EDTA. Significant differences of at least Pb0.05 (Wilcoxon rank test) between the samples are indicated by the following symbols: (a) from serum ; (b) from serum+; (c) from plasmacitrate; (d) from plasma-heparin; (e) from plasma-EDTA.
2
Letter to the Editor
white blood cell count are observed during the disease activity and the treatment of multiple sclerosis and could subsequently result in changed TIMP-1 concentrations when measurements are performed in serum. In summary, these important preanalytical conditions should be considered in the interpretation of changed MMP9 and TIMP-1 levels. Recently, the use of blood samples collected with sodium citrate was suggested to be the best procedure to avoid the detrimental effect of other anticoagulants or serum and to optimize the diagnostic validity of MMP-9 in peripheral blood (Mannello et al., 2003).
References Blanco, Y., Saiz, A., Carreras, E., Graus, F., 2004. Changes of matrix metalloproteinase-9 and its tissue inhibitor (TIMP-1) after autologous hematopoietic stem cell transplantation in multiple sclerosis. J. Neuroimmunol. 153, 190 – 194. Cooper, T.W., Eisen, A.Z., Stricklin, G.P., Welgus, H.G., 1985. Plateletderived collagenase inhibitor: characterization and subcellular localization. Proc. Natl. Acad. Sci. U. S. A. 82, 2779 – 2783. Jung, K., Nowak, L., Lein, M., Henke, W., Schnorr, D., Loening, S.A., 1996. What kind of specimen should be selected for determining tissue
inhibitor of metalloproteinase-1 (TIMP-1) in blood? Clin. Chim. Acta 254, 97 – 100. Jung, K., Laube, C., Lein, M., Lichtinghagen, R., Tschesche, H., Schnorr, D., Loening, S., 1998. Kind of sample as preanalytical determinant of matrix metalloproteinases 2 and 9 (MMP2; MMP9) and tissue inhibitor of metalloproteinase 2 (TIMP2) in blood. Clin. Chem. 44, 1060 – 1062. Kodama, S., Iwata, K., Iwata, H., Yamashita, K., Hayakawa, T., 1990. Rapid one-step sandwich enzyme immunoassay for tissue inhibitor of metalloproteinases. An application for rheumatoid arthritis serum and plasma. J. Immunol. Methods 127, 103 – 108. Mannello, F., Luchetti, F., Canonico, B., Papa, S., 2003. Effect of anticoagulants and cell separation media as preanalytical determinants on zymographic analysis of plasma matrix metalloproteinases. Clin. Chem. 49, 1956 – 1957.
Klaus Jung Department of Urology, University Hospital Charite´, Humboldt University Berlin, Schumannstrasse 20/21, D-10098 Berlin, Germany E-mail address: [email protected] Tel.: +49 30 450 515041/515127; fax: +49 30 450 515904. 9 December 2004