INFLUENCE OF BLOOD SAMPLING PROCEDURE ON PLASMA CONCENTRATIONS OF MATRIX METALLOPROTEINASES AND THEIR TISSUE INHIBITORS

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Abstract
1. Plasma levels of matrix metalloproteinases (MMP) and tissue inhibitors of metalloproteinases (TIMP) are potential markers of many diseases involving extracellular matrix remodelling such as hypertension. Our aim was to determine whether the anticoagulant used to collect plasma and several freeze-thaw cycles may influence the accuracy of plasma MMP and TIMP determinations. 2. Plasma samples of 18 healthy volunteers were collected on three anticoagulants: heparinate, citrate and EDTA. For each anticoagulant, we compared: (i) MMP-2 and MMP-9 levels using gelatin zymography and TIMP-1 and TIMP-2 concentrations using enzyme-linked immunosorbent assay; (ii) intra- and interassay coefficients of variation (CV); and (iii) MMP and TIMP levels after up to five freeze-thaw cycles. 3. The choice of anticoagulant influenced TIMP-2 and TIMP-1 concentrations (TIMP-2, P < 0.0001; paired comparisons, citrate vs EDTA, P < 0.0001; EDTA vs heparin, P < 0.0001; citrate vs heparin, P < 0.0001; TIMP-1, P < 0.001; paired comparisons, citrate vs EDTA, P = 0.10; EDTA vs heparin, P < 0.01; citrate vs heparin, P < 0.0001), but not those of MMP. We observed a bias with heparinate for TIMP-2, TIMP-1 and MMP-9 determinations. The anticoagulant did not influence intra-assay or interassay CV. Performing freeze-thaw cycles led to alterations in the TIMP-1 plasma levels (P < 0.0001), regardless of the anticoagulant used, whereas MMP and TIMP-2 concentrations were not significantly affected. 4. Anticoagulant influences the measured levels of MMP and TIMP in plasma and should be systematically reported. However, it does not influence the reproducibility of the measurements. Repeated freeze-thaw cycles alter the measurement of TIMP-1 levels and should be avoided.