Levels of matrix metalloproteinases differ in plasma and serum – aspects regarding analysis of biological markers in cancer

SHORT COMMUNICATION British Journal of Cancer (2016) 115, 703–706 | doi: 10.1038/bjc.2016.127 Keywords: Colorectal cancer; neoplasm; biological markers; matrix metalloproteinases; MMPs; blood plasma; blood serum Levels of matrix metalloproteinases differ in plasma and serum – aspects regarding analysis of biological markers in cancer Andreas Jonsson1,2, Claes Hjalmarsson3, Peter Falk2 and Marie-Lois Ivarsson*,1,2 1 Hallands Hospital Varberg, Region Halland, SE-432 37 Varberg, Sweden; 2Fibrinolysis Laboratory, Department of Surgery, Institute of Clinical Sciences, Sahlgrenska Academy at the University of Gothenburg, SE-416 85, Göteborg, Sweden and 3 Department of Surgery, Kalmar Hospital, SE-391 26 Kalmar, Sweden Background: There are inconsistencies in the use of serum or plasma when analysing the matrix metalloproteinases (MMPs) as diagnostic or prognostic markers. The purpose of this study was to compare the concentration of MMP-1, -2, -7, -8, -9 and -13 in serum vs plasma samples. Methods: Blood samples were obtained from sixty-five men and women. Samples were analysed for levels of MMPs in corresponding citrate plasma and serum. Results: All MMPs expressed higher concentration in serum compared with plasma (Po0.01). There were no differences between genders. Conclusions: Present study demonstrated significant differences regarding concentrations of some MMPs using plasma vs serum. We conclude that future studies regarding MMPs as biological markers in cancer should consider the use of citrate plasma instead of serum. Matrix metalloproteinases (MMPs) are proteases that have a major role in the degradation of the extracellular matrix (Langenskiold et al, 2005). MMPs can be synthesised not only by the tumour cell, but also from surrounding stromal cells. The activity and expression of MMPs is increased in many types of cancer (Cauwe et al, 2007). Numerous studies indicate a possible value of MMP concentration in blood samples from patients with colorectal cancer, either as a diagnostic or prognostic marker (Tutton et al, 2003; Maurel et al, 2007; Kushlinskii et al, 2013). There is however a lack of consensus with data pointing at different directions and inconsistencies in using serum or plasma analysing MMPs in patients with colorectal neoplasia (Hurst et al, 2007; Damery et al, 2013). Analysing serum for determination of circulating MMP has previously been criticised (Gerlach et al, 2005). MMPs releases from leucocytes during the clotting process, which makes serum samples time-to-analyse dependent (Zucker and Cao, 2005). As a part of further investigation of the MMPs and their use as a diagnostic and prognostic marker, we have explored differences between plasma and serum levels of MMP. The aim of this study was to compare the concentration of MMP-1, -2, -7, -8, -9 and -13 in serum and plasma samples for each patient in a population suitable for a colon cancer-screening programme. MATERIALS AND METHODS Blood collection and sample preparation. Venous blood samples were collected in a standardised way from 65 (34 males, 31 females), 65-years-old individuals participating in a study regarding colorectal cancer screening. Serum samples were collected in tubes without clot activators; plasma samples were collected in citrate tubes. Serum tubes were stored in room temperature ( þ 20 1C) for 30 min and then centrifuged at 10 000 g in 20 1C, citrate tubes were centrifuged within 5 min at 10 000 g in 20 1C. *Correspondence: Professor M-L Ivarsson; E-mail: Received 16 February 2016; revised 7 April 2016; accepted 20 April 2016; published online 17 May 2016 & 2016 Cancer Research UK. All rights reserved 0007 – 0920/16 www.bjcancer.com | DOI:10.1038/bjc.2016.127 703 BRITISH JOURNAL OF CANCER Protein level measurement. In all samples levels of MMP-1, MMP-2, MMP-7, MMP-8, MMP-9 and MMP-13 were analysed with the Luminex xMap multi-assay technology (Bio-Plex 200, BIO-RAD, Sundbyberg, Sweden). The samples were measured simultaneously with Fluorokine MAP (Multi-Analyte Profiling, RnD Systems, Abingdon, UK) assay according to the manufacturer’s instructions. For MMP-1, the kit detects pro-, mature and TIMP-1 complex MMP-1, with a lower detection limit of 0.57 pg ml  1 together with an intra assay (CV%) of 7.8–9.0 and an inter-assay of 15.3–16.2%, respectively. For MMP-2, the kit detects pro- and mature MMP-2, with a lower detection limit of 3.8 pg ml  1, and an intra assay (CV%) of 7.3–9.3 and an interassay of 10.0–13.3%, respectively. For MMP-7, the kit detects pro-, mature and TIMP-1-complexed MMP-7, with a lower detection limit of 3.9 pg ml  1 together with an intra assay (CV%) of 5.0–9.0 and an inter-assay of 7.7–11.5%, respectively. For MMP-8, the kit detects pro-, mature and TIMP-1-complexed MMP-8, with a lower detection limit of 7.8 pg ml  1 together with an intra assay (CV%) of 5.2–7.0 and an inter-assay of 9.6–14.3%, respectively. For MMP-9, the kit detects pro- and mature MMP-9, with a lower detection limit of 5.7 pg ml  1 together with an intra assay (CV%) of 3.8–5.8 and an inter-assay of 9.3–11.7%, respectively. For MMP13, the kit detects pro-, mature and TIMP-1 complexed MMP-13, with a lower detection limit of 36.5 pg ml  1 together with an intra assay (CV%) of 4.3–5.6 and an inter-assay of 10.7–12.6%, respectively. All samples were prediluted 11-fold. Each sample was measured in duplicates and protein levels in the samples were calculated using a five parameter logistic (5-PL) standard curve according to the manufacture instructions. Statistical analysis. The MMP analysis was transformed with the natural logarithm to get normally distributed data; this was possible for MMP-1, MMP-8 and MMP-9. Dependent t-test was used to compare means between citrate plasma and serum samples. For data not normally distributed the Wilcoxons-signed rank test was used to compare medians (MMP-2, MMP-7). The Mann– Whitney U test was used to test for differences between genders in the non-normally distributed data, and the independent t-test was used for normally distributed data. Correlation between serum and plasma levels was estimated using the Spearman-rank correlation test. All test were two-sided. Po0.05 was considered significant. All calculations were carried out with IBM SPSS Statistics for Macintosh (Ver. 22.0, IBM Corp, Armonk, NY, USA). Ethics. Informed consent was obtained from all participating patients. The study has approval from the Local Ethics Committee at Lund University Hospital, Lund, Sweden. RESULTS In all assays the median levels of the different MMPs found in serum generated a higher value than corresponding levels in citrated plasma (Po0.01). Median and ranges for each assay are reported in detail in Table 1. Levels of MMP-13 were not presented due to concentrations below the detection level. When comparing the distribution of MMP levels it was found that the interquartile ranges (IQR) were all greater in the serum samples compared to parallel (...truncated)