Kind of Sample as Preanalytical Determinant of Matrix Metalloproteinases 2 and 9 and Tissue Inhibitor of Metalloproteinase 2 in Blood

Matrix metalloproteinases (MMPs) and tissue inhibitors of metalloproteinases (TIMPs) are interesting new diagnostic tools in oncology, liver diseases, and rheumatoid arthritis (1)(2)(3)(4)(5). However, the preanalytical issue of their determination in blood was only recently shown (6). We have continued our investigations concerning MMP-2, MMP-9, and TIMP-2 because commercial ELISA assays are only now available. We will shortly report the results and present some conclusions. The study design corresponded to the approach described previously (6). Briefly, we performed BIOTRAK™ ELISA assays for MMP-2, MMP-9, and TIMP-2 (Amersham). Blood samples from nine healthy male volunteers were simultaneously collected into plastic tubes for preparation of serum samples and into potassium EDTA-coated tubes as well as into lithium heparin-coated plastic tubes for preparation of plasma samples (Monovette systems 03.1528, 05.1167, and 03.1589; Sarstedt). The tubes stored at room temperature were centrifuged within 30 min after venipuncture at 1600g for 15 min at 4 °C. The supernatants were stored at −80 °C until analysis. Fig. 1 , A–C, shows that MMP-2, MMP-9, and TIMP-2 measured in serum, heparin plasma, and EDTA plasma are markedly different. Although MMP-2 values did not differ in serum and heparin plasma but were lower in EDTA plasma, the MMP-9 concentration was ∼3- to 20-fold higher in serum than in heparin and EDTA plasma, respectively. In contrast, TIMP-2 values were five- to eightfold higher in heparin plasma than in serum and EDTA plasma. To realize the possible inhibitory or stimulatory effect of EDTA and heparin on the determination of these components, we added EDTA or heparin to serum samples (n = 4), getting concentrations equivalent to those in the mentioned monovette systems (2 g EDTA or 30 000 IU heparin/L sample). MMP-2 and MMP-9 concentrations were 96–106% of the initial values without additives, demonstrating no interference by EDTA and heparin. However, heparin but not EDTA produced increased TIMP-2 values dependent on the concentration but not on the kind of heparin (Fig. 1D). (A) MMP-2, (B) MMP-9, and (C) TIMP-2 concentrations in dependence on sample processing; (D) the influence of heparin on the TIMP-2 measurement. (A–C) The analytes were measured in serum and plasma derived from blood samples of nine healthy men collected either in EDTA- or lithium heparin-coated tubes. Individual values and the medians are given. Significance levels were calculated by the Wilcoxon rank test for paired data (at least P <0.05): a, significant difference between serum and heparin plasma; b, significant difference between serum and EDTA plasma; and c, significant difference between heparin plasma and EDTA plasma. (D) TIMP-2 concentrations in serum samples (n = 4) were measured after the addition of different concentrations of lithium, potassium, and ammonium heparin. Data are given as percentages of the arithmetic means ± SD of the samples without heparin, indicated as 100%. We conclude from these data that differences of MMP-2 and MMP-9 measured in serum and plasma result from the sampling process, whereas variations of TIMP-2 between serum and heparin plasma are directly affected by heparin. These results are of special interest because MMP-2, MMP-9, and TIMP-2 were measured both in serum and in plasma samples, and different data were presented (1)(2)(3)(4)(5)(7)(8)(9). To assess these discrepancies, several reasons have to be considered. There is no doubt that general points, such as lack of a common calibration material and the matrix effects, are most important when different tests are used. Sparse information exists on how specimen collection affects the variation of those components. No data are available on whether the differences observed in various samples are possibly caused by an additional release of these components from blood cells (e.g., from platelets during platelet activation) (10) and/or by the occurrence/prevention of activation of MMPs by the special sampling process. The decisive factor is whether only the latent (proforms) or also the active forms of MMPs are detected (11)(12). The corresponding monoclonal antibodies for their detection are available (12)(13). For the determination of MMP-2, several studies used the ELISA test of Fujimoto et al. (14). This test measured free proMMP-2 and proMMP-2-TIMP-2 complexes but not active MMP-2. The upper reference limit of serum MMP-2 obtained with this test was 920 μg/L (7). Using the Amersham test kit based on the method of Fujimoto et al. (14), we found in heparin plasma of 40 healthy women an upper 95% limit of 985 μg/L. Another ELISA test of MMP-2 (15) detects latent and activated MMP-2, MMP-2 complexed with TIMP, and α2-macroglobulin. The authors (1) recommended measuring MMP-2 in EDTA plasma because serum concentrations were considerably higher, but they found a comparable upper cutoff of 828 μg/L. Because our data showed that EDTA plasma reduces MMP-2 concentrations by a factor of ∼5 compared with serum, we believe that the apparent similar cutoffs do not prove that the tests measure similar components. A similar conclusion must be drawn for the determination of MMP-9. Fujimoto et al. (16) developed an ELISA that measures free proMMP-9, proMMP-9-TIMP1 complexes, and active 83-kDa MMP-9 but not active 67-kDa MMP-9. A 95% central reference interval between 10–63 μg/L was found in plasma (3). That test has also been applied both to serum and plasma samples (2). The ELISA for MMP-9 described by Bergmann et al. (12) detects both proMMP-9 and the activated forms. Consequently, a higher upper cutoff limit (90% range) of 94 μg/L was given. A few studies reported on TIMP-2 concentrations in blood. Until now, only serum concentrations have been given, with mean values of about 36–74 μg/L (2)(5)(8). The one-step sandwich ELISA of Fujimoto et al. (17), which is also the basis of the assay used in our study, measures free TIMP-2 and TIMP-2 complexes with the active forms of MMP-1, -2, -3, and -9 but not TIMP-2 complexed with proMMP-2. This special design may explain the effect of heparin on the determination of TIMP-2, because heparin might interact with proMMP-2 in the proMMP-2-TIMP-2 complex, as found in studies on interaction between heparin and collagenase (18). Released TIMP-2 from the proMMP-2-TIMP-2 complex could cause the measurement of increased free TIMP-2, as shown in our experiments with increasing concentrations of heparin. Whatever the definitive mechanism for that effect, heparin plasma seems to be an inappropriate sample for determining circulating TIMP-2 in blood. In conclusion, the clinician should be aware that the commutability of MMP and TIMP values measured by ELISAs are impossible if different kinds of specimens are used. To avoid misinterpretations, at least consistent specimens should be used. However, we fear that an inappropriate specimen affects the diagnostic validity of these markers. Thus, there is an urgent need to solve the possibly mutual problems of the proper specimen for the determination of MMPs and TIMPs and whether the proforms, active, or total forms of MMPs should be measured. This work includes parts of the doctoral thesis of C.L. and was supported in part from the Fonds der Chemischen Industrie (K.J., project no. 400700) and Deutschen Forschungsgemeinschaft (Ju 365/3–1). We thank Silke Klotzek for valuable technical assistance. and *address for correspondence: Department of Urology, University Hospital Charité Humboldt University, Schumannstraβe 20/21, D-10098 Berlin, Germany fax 4930 28021402, e-mail jung@rz.charite.hu-berlin.de