Blood sampling as critical preanalytical determinant to use circulating MMP and TIMP as surrogate markers for pathological processes

Abstract

Dear Sir,It has been suggested that circulating matrix metalloproteinases (MMP) and tissue inhibitors of matrix metalloproteinases (TIMP) may be useful diagnostic and prognostic indicators in malignant and nonmalignant diseases.1 Numerous reports seem to confirm that assumption although it is unknown whether increased MMP concentration are based on the initial step of tissue damage or the following repairing process.2 There are also contrasting results when similar patients were studied, i.e., increased plasma MMP-9 concentrations but unchanged serum concentrations were found in patients with colon carcinoma. 3,4 We believe that in addition to the analytical and clinical reasons to explain these differences between various studies, more attention should be given to the preanalytical conditions of sampling. In this respect, we read with great interest a recent article published in this journal.5 Gianelli et al. 5 found no difference in serum concentrations of MMP-9 and TIMP-1 between healthy women and patients with breast cancer, but decreased MMP-9 and increased TIMP-1 after surgery in comparison with initial concentrations. The authors concluded that these changes might serve as prognostic indices in the follow-up of breast cancer patients. It should be remembered that, however, that using serum material as samples for the determination of those analytes, the authors had rather inadequately considered the impact of blood collection as an important preanalytical determinant of MMP and TIMP results. There is rising evidence that blood sampling and handling markedly determine the concentrations of circulating MMP and TIMP.6-12 Thus, to interpret that data correctly and to avoid wrong expectations in future studies using these markers, we want to draw the attention of interested clinicians to these facts that were particularly discussed in analytical journals. [6][7][8]12 Although the pitfalls in the measurement of circulating growth factors and cytokines for diagnostic purposes have become generally accepted, 13 similar evidences for MMP and TIMP have been widely ignored.Results of own experiments (summarized in Fig. 1) demonstrate the differences of MMP-9 and TIMP-1 in serum and plasma as the analytes come into question. Briefly, for MMP-9 measurements, blood samples from 12 healthy volunteers were collected in Vacutainer tubes (Becton Dickinson, Franklin Lakes, NJ) containing a clot activator for serum preparations or lithium heparinate, dipotassium EDTA or sodium citrate for plasma preparations. Corresponding prepared plastic tubes (Monovettes; Sarstedt, N€ urtingen, Germany) were used for blood sampling for TIMP-1 measurements. For the MMP-9 experiments, the tubes were either centrifuged (1,600g, 15 min) immediately (considered as t 0 ) or after 0.5 or 2 hr storage at room temperature and after 24 hr storage at 4°C, respectively. Within 5 days of sampling while the samples were stored at 4°C MMP-9 measurements were carried out using our own enzyme immunoassay described previously.14 Briefly, samples (and sta...

Soluble interleukin-2 receptor and metalloproteinase-9 expression in head and neck cancer: prognostic value and analysis of their relationships

“…As recommended, we have measured MMP-9 in plasma and not in serum, because MMP-9 measurement in serum reflects primarily release of proteases by leucocytes during the clotting process in the blood collection tube [56,57]. Our tubes were stored at -80°C before MMP-9 assay.…”

Blood sampling affects circulating TIMP‐1 concentration, a useful biomarker in estimating liver fibrosis stages†

“…1). [5][6][7] In fact, platelets and leukocytes contain several TIMPs that may be extracellularly released on activation or during aggregation, and the use of anticoagulant (for example, heparin, citrate, and ethylene diamine tetraacetic acid [EDTA]) limits the mobilization of granules from human neutrophils. [4][5][6] The presence of TIMPs in higher amounts in serum than in plasma 5-8 is related not only to disease status but also to releasing mechanism(s) of coagulation/fibrinolytic pathways.…”

“…[5][6][7] In fact, platelets and leukocytes contain several TIMPs that may be extracellularly released on activation or during aggregation, and the use of anticoagulant (for example, heparin, citrate, and ethylene diamine tetraacetic acid [EDTA]) limits the mobilization of granules from human neutrophils. [4][5][6] The presence of TIMPs in higher amounts in serum than in plasma 5-8 is related not only to disease status but also to releasing mechanism(s) of coagulation/fibrinolytic pathways. 4,9 The individual differences in white blood cell and platelet count, the coagulation/fibrinolysis factors, 4,9 and the sampling and handling procedures of blood collection (presence of clot accelerators in serum collection tubes 4,6 and handling temperature before and during centrifugation 8 ) significantly alter the concentrations of TIMPs 5-8 comparable with modifications in protein profiles observed in proteomic studies.…”

“…[4][5][6] The presence of TIMPs in higher amounts in serum than in plasma 5-8 is related not only to disease status but also to releasing mechanism(s) of coagulation/fibrinolytic pathways. 4,9 The individual differences in white blood cell and platelet count, the coagulation/fibrinolysis factors, 4,9 and the sampling and handling procedures of blood collection (presence of clot accelerators in serum collection tubes 4,6 and handling temperature before and during centrifugation 8 ) significantly alter the concentrations of TIMPs 5-8 comparable with modifications in protein profiles observed in proteomic studies. 10 All these data add to the mounting evidence that serum does not seem to be an appropriate sample for determining circulating TIMP-1, whereas plasma represents the optimal choice to better evaluate TIMPs for clinical and diagnostic purposes, 9 especially to evaluate disease stage information in liver diseases such as the presence or absence of advanced fibrosis or cirrhosis.…”

A strong note of caution in using matrix metalloproteinase‐1 and its inhibitor, TIMP‐1 in serum as biomarkers in systolic heart failure

“…Both variables are critical determinants that affect the concentration levels of MMPs and TIMPs [9][10][11]. Disregarding these facts would be unsatisfactory for the accurate measurement of circulating MMPs and TIMPs and it is strongly recommended to consider these possible pitfalls to avoid misinterpretation of results.…”