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Preanalytical Biases in Measurement of Matrix Metalloproteinases and Their Tissue Inhibitors in Peripheral Blood

To the Editor:

Fiedorczyk, et al¹ recently analyzed serum concentrations of the matrix metalloproteinases (MMP) MMP-1, MMP-3, MMP-9, and MMP-13 and their tissue inhibitors (TIMP) TIMP-1 and TIMP-2 in patients with early rheumatoid arthritis (RA) before and after treatment with methotrexate. They found elevated concentrations in comparison with patients with osteoarthritis and reduced levels and ratios of MMP to TIMP after therapy. Since these results strongly correlated with clinical signs of disease activity and laboratory data such as C-reactive protein, the authors concluded that the determination of serum MMP and TIMP would be helpful to characterize the disease activity of RA. In previous reports, the authors also used serum as samples for the measurements of MMP and TIMP^2,3, but did not clearly describe what type of collection system, either with or without clot activator, they used. On the other hand, the influence of the kind of blood sample, either collected as serum with or without clot activator or as plasma with different types of anticoagulants like heparin, citrate, or EDTA, on the measurement of these analytes was discussed in detail in analytical and clinical journals^4-9. As yet no information on the effect of blood sampling on circulating MMP-1 and MMP-3 is available in the literature. However, since the reasons for that effect do not seem to be restricted to only MMP-9 and TIMP-1, I believe the clinical readership should be aware of that general problem. I should like to demonstrate it by summarizing some of my own experiments.

Briefly, blood samples from 8–10 healthy adults were simultaneously prepared in plastic tubes (Monovette Systems, Sarstedt GmbH, Nümbrecht, Germany) without additives or with kaolin-coated plastic granulate as coagulation accelerator to prepare native serum or serum after enhanced coagulation and in respective devices coated with lithium heparin to obtain plasma. Within 30 min after venipuncture, the blood samples were centrifuged at 1600 ´ g for 15 min; the collected supernatants were recentrifuged and stored at –80°C until analysis. ELISA kits from Amersham (Little Chalfont, Buckinghamshire, UK) were used for the measurements of MMP-1, MMP-3, TIMP-1, and TIMP-2; MMP-9 was determined with a kit from Medac Diagnostika, Wedel, Germany.

Data are summarized in Figure 1. The following conclusions can be drawn: (1) Concentrations of MMP-1, MMP-3, MMP-9, and TIMP-1 are several-fold higher in serum than in plasma (means of 6 for MMP-1, 2.3 for MMP-3, 5.3 and 20.3 for MMP-9, and 6.9 for TIMP-1). (2) Serum samples collected with clot activator as the conventional way to prepare serum for routine use show higher concentrations of MMP, for example, MMP-9. (3) In contrast, TIMP-2 concentrations are about 9-fold higher measured in heparin plasma than in serum.

[click, then close, image]

Figure 1. Concentrations of MMP-1, MMP-3, MMP-9, TIMP-1, and TIMP-2 in serum and plasma samples. Plastic tubes without additives or with kaolin-coated plastic granulate as coagulation accelerator were used to prepare native serum (serum–) and serum after enhanced coagulation (serum+); tubes coated with lithium heparin were used to obtain plasma.

The reason for these different concentrations of MMP-1, MMP-3, MMP-9, and TIMP-1 in serum and plasma is very likely the variable release of these components from platelets and leukocytes depending on the different blood collection procedures, as these blood cells contain high concentrations of these analytes⁷. TIMP-2 increased with increasing heparin concentration, probably because of the interaction of heparin with the proMMP-2-TIMP-2-complex⁵.

These data illustrate the problem: to measure true concentrations of MMP and TIMP in peripheral blood and to come to trustworthy conclusions with samples collected under different conditions. Recently, citrate has been suggested to be the best anticoagulant to prepare blood samples for measuring MMP-2 and MMP-9 in peripheral blood^6,8, while recommendations for TIMP and other MMP have not been provided yet. However, the high unspecific background found in serum due to the release of the MMP or TIMP from blood cells during sample preparation may hamper the actual diagnostic or prognostic information of MMP and TIMP. Since white blood cell count and release of the analytes from blood cells may additionally change during treatment¹⁰, the usefulness of MMP and TIMP as monitoring markers may be rather limited, despite a consistent use of serum collected under identical conditions.

Thus, the clinician should be aware of these preanalytical pitfalls to avoid misinterpretation of data when MMP and TIMP should be used for clinical purposes.

KLAUS JUNG, MD, Department of Urology, Charité—Universitätsmedizin Berlin, Schumannstrasse 20/21, D-10117 Berlin, Germany. E-mail: klaus.jung@charite.de

REFERENCES

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9. Souza-Tarla CD, Uzuelli JA, Machado AA, Gerlach RF, Tanus-Santos JE. Methodological issues affecting the determination of plasma matrix metalloproteinase (MMP)-2 and MMP-9 activities. Clin Biochem 2005;38:410-4.

10. Seitz M, Dayer JM. Enhanced production of tissue inhibitor of metalloproteinases by peripheral blood mononuclear cells of rheumatoid arthritis patients responding to methotrexate treatment. Rheumatology Oxford 2000;39:637-45.