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


  • Ferdinando Mannello Ph.D.,

    1. Institute of Histology and Laboratory Analysis, Faculty of Sciences and Technologies, University “Carlo Bo”, Urbino, Italy
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  • Klaus Jung M.D.

    1. Department of Urology, University Hospital Charité, Humboldt University; and Berlin Institute for Urologic Research, Berlin, Germany
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  • Potential conflict of interest: Nothing to report.

Blood Sampling Affects Circulating TIMP-1 Concentration, a Useful Biomarker in Estimating Liver Fibrosis Stages

To the Editor:

Liver fibrosis and its end-stage disease cirrhosis are a major cause of mortality and morbidity throughout the world. Several serum biomarkers and panels offer the opportunity to assess the extent of liver disease noninvasively, but only a few of the noninvasive serum markers allow the determination of different stages of fibrosis on a continuum similar to that achieved with liver biopsy.1

In this respect, Dr. Fontana and colleagues2 reported an excellent and interesting study about the utility of a new panel of serum fibrosis markers (platelet count, hyaluronic acid, and tissue inhibitor of matrix metalloproteinase-1 [TIMP-1]) which shows a stronger correlation with Ishak scores to reflect the pattern of fibrosis more closely than does the quantity of hepatic collagen. Because regression of liver fibrosis is mediated by decreased expression of TIMP,3 modified concentrations of circulating TIMP-1 could reflect the alterations in tissue matrix remodelling during hepatic fibrogenesis and fibrolysis. However, according to growing literature evidence demonstrating that blood sampling strongly influences the measurement and recovery of true circulating matrix metalloproteinases (MMPs) and TIMPs (reviewed by Mannello4), we would like to draw attention to the preanalytical impact of blood collection methods in order to limit technical pitfalls that may lead to misinterpretations.

The discrepancy in both MMP and TIMP concentrations and zymographic profiles between serum and plasma has been extensively demonstrated.4 In particular, serum samples were found to contain higher levels of TIMP-1 than those in plasma (Fig. 1).5–7 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.4–6 The presence of TIMPs in higher amounts in serum than in plasma5–8 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 tubes4, 6 and handling temperature before and during centrifugation8) significantly alter the concentrations of TIMPs5–8 comparable with modifications in protein profiles observed in proteomic studies.10

Figure 1.

Effects of blood sampling on the concentrations of TIMP-1. We analyzed 30 healthy volunteers aged 23-55 years (median 36 years). Samples were collected in plastic tubes (Vacutainer; Becton Dickinson, Franklin Lakes, NJ). Serum: pure serum prepared in plastic tubes without additives; Serum + C.A.: serum prepared in plastic tubes containing silica particles as clot activators. Plasma was prepared in tubes coated with lithium heparin or potassium EDTA (Heparin and EDTA, respectively). After centrifugation at 1500g for 12 minutes at 4°C, the supernatants of samples were analyzed for TIMP-1 concentration by commercially available enzyme-linked immunosorbent assay kit (Biotrak ELISA kit, RPN 2611; Amersham Pharmacia). Columns, mean; Bars, standard errors. Statistical analyses were performed with Prism software (GraphPad Software, San Diego, CA). Differences were compared using a paired t test. In all instances, significance was set at P < 0.05. Significant differences between samples were indicated.

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. The high unspecific background found in serum may hamper the actual diagnostic/prognostic information of TIMPs. To avoid pitfalls that may lead to false conclusions or expectations, it is important that investigators or clinicians who use blood TIMP as a surrogate biomarker in determination of liver fibrosis/cirrhosis be cognizant of the crucial impact of blood sampling and handling that may undermine confidence in the conclusions of an otherwise well-conceived study.3

Thus, to obtain reproducible and comparable TIMP-1 values in clinical studies,8 standardized operating procedures for sample handling are necessary to avoid misinterpretations.4

Ferdinando Mannello Ph.D.*, Klaus Jung M.D.†, * Institute of Histology and Laboratory Analysis, Faculty of Sciences and Technologies, University “Carlo Bo”, Urbino, Italy, † Department of Urology, University Hospital Charité, Humboldt University; and Berlin Institute for Urologic Research, Berlin, Germany.