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1 Istituto di Istologia ed Analisi di Laboratorio,
2
Istituto di Scienze Morfologiche, and,
3
Centro di Citometria e Citomorfologia, Università degli Studi "Carlo Bo", 61029 Urbino PU, Italy
aAuthor for correspondence. Fax 39-0722-322370; e-mail f.mannello{at}uniurb.it
To the Editor:
Matrix metalloproteinases (MMPs) are calcium/zinc-dependent endoproteinases involved in physiologic and pathologic processes, modulating extracellular matrix degradation (1). MMP-2 (EC 3.4.24.24) and MMP-9 (EC 3.4.24.35) circulating in the peripheral blood (PB) of patients with neoplasia showed contrasting results, revealing the preanalytical issue of whether the method of PB sampling influences MMP concentrations (2) and their zymographic profiles (3). We therefore analyzed the effects of anticoagulants and cell separation media on PB gelatinolytic profiles.
PB samples from 30 healthy volunteers were collected into VacutainerTM Tubes with clot activator (SST), lithium heparin (LH), dipotassium EDTA (K2E), sodium fluoride/potassium oxalate (NaF/KOx), and buffered/acidic citrate [natrium citrate (9NC), acid-citrate-dextrose (ACD), and citrate-phosphate-dextrose-adenine (CPDA); Becton Dickinson]. After centrifugation at 500g for 15 min at 4 °C, the supernatants and buffy coats were collected and analyzed. Leukocyte subpopulations were obtained after Lympholyte® gradient (5.64% NycogradeTM Polysucrose 400, 9.65% sodium diatrizoate; Cedarlane), and their subset recovery was tested through cytometric analysis (4). Gelatinases from leukocytes and plasma samples were analyzed by gelatin zymography, with 150 µg of total protein loaded on the gel (3). Calibrators were prepared from capillary PB (5). MMP-2 and MMP-9 were measured by ELISA (5). Differences were compared using the Mann–Whitney U-test and the paired t-test; P values <0.05 were statistically significant.
The present work was carried out in accordance with the ethics standards of the Helsinki Declaration of 1975, as revised in 1983.
We found in plasma a 72-kDa constitutive gelatinase that was produced by nonproteolytic activation of MMP-2 with sodium dodecyl sulfate, and additional MMP-9 forms at 92, 130, and 225 kDa. Western blot analysis, Ca2+/Zn2+ dependence, and p-aminophenyl-mercuric acetate activation (data not shown) identified the plasma gelatinases as fibroblast-derived proMMP-2 and neutrophil-derived proMMP-9, circulating as latent activatable proenzymes.
MMP-9 was significantly higher in serum than in citrate plasma [mean (SD), 59 (7) vs 8 (2) µg/L, respectively; P <0.01], but the active/latent isoform proportions were similar (Fig. 1A
). Platelet aggregation during clotting (6) may have caused these differences.
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MMP expression was lower in K2E plasma than in LH plasma [14 (3) vs 25 (4) µg/L; P <0.05; Fig. 1A
, lane 4 vs lane 5]. The concentrations of MMP-9 forms decreased significantly with increasing amounts of K2E during PB collection, whereas MMP-2 was increased (P <0.01; Fig. 1B
). When we added anticoagulants to the zymography buffer (to mimic the conditions in Vacutainer Tubes), only K2E inhibited the gelatinolytic activities (data not shown). Although EDTA may alter MMP expression (7), the reasons for the contrasting K2E effects remain unknown.
To minimize interindividual variability, we collected PB from the same individual into different buffers. We found mainly proMMP-2 in the buffered/acidic citrate plasma [202 (15) µg/L], whereas there were no statistically significant differences among the 9NC, ACD, and CPDA plasmas. We found additional proMMP-9 in the K2E, LH, and NaF/KOx plasmas (Fig. 1A
, lanes 1–3 vs lanes 4–6). Our observations revealed that anticoagulants can act as preanalytical determinants of PB MMPs.
LH and 9NC plasmas collected after Lympholyte gradient (Fig. 1C
, lanes 3 and 4 vs lanes 1 and 2), as well as after 9.65% sodium diatrizoate alone (Fig. 1C
, lanes 5 and 6 vs lanes 3 and 4), showed increased concentrations of all MMPs. Polysucrose 400 alone did not affect MMP concentrations (data not shown) or isoform profiles.
Cytometric analysis revealed differences in MMP composition between leukocytes from LH PB vs leukocytes from 9NC PB (data not shown). Physiologic buffy coats from 9NC PB showed only MMP-9 forms, had lower gelatinase activity, and had a different zymographic profile with respect to LH PB (Fig. 1C
, lane 7 vs lane 8). The MMP differences between LH vs 9NC plasma could be caused by differential release of MMPs from, e.g., platelets and leukocytes, with a changed MMP content/profile depending on the anticoagulant used (6)(8).
Although previous reports suggested heparin as the anticoagulant of choice to study circulating MMPs (2)(3), to optimize the diagnostic validity of PB MMPs as cancer biomarkers (1), we recommend the use of buffered/acidic citrate (9NC, ACD, and CPDA), whereas LH, K2E, and NaF/KOx, which affect the MMP content and zymographic profiles of plasma and leukocytes, should be avoided.
References
The following articles in journals at HighWire Press have cited this article:
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  F. Mannello Serum or Plasma Samples?: The "Cinderella" Role of Blood Collection Procedures Preanalytical Methodological Issues Influence the Release and Activity of Circulating Matrix Metalloproteinases and Their Tissue Inhibitors, Hampering Diagnostic Trueness and Leading to Misinterpretation Arterioscler. Thromb. Vasc. Biol., April 1, 2008; 28(4): 611 - 614. [Full Text] [PDF]
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K. Jung Matrix Metalloproteinase-8 and Tissue Inhibitor of Metalloproteinase-1 in Serum Do Not Reflect the Analytes Circulating in Blood Arterioscler. Thromb. Vasc. Biol., March 1, 2008; 28(3): e15 - e16. [Full Text] [PDF]
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 F. Mannello, G. A. Tonti, J. E. Tanus-Santos, and R. F. Gerlach Silicate Increases the Release of MMP-9 Forms in Peripheral Blood: Why Gelatin Zymography Differs Significantly in Citrate Plasma and Serum Obtained with or without Clot Activators Clin. Chem., November 1, 2007; 53(11): 1981 - 1982. [Full Text] [PDF]
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 E. Fainardi, M. Castellazzi, T. Bellini, and F. Dallocchio Matrix metalloproteinases (MMP): determination of different forms by different techniques may require different preanalytical strategies. Multiple Sclerosis, June 1, 2007; 13(5): 691 - 692. [PDF]
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F. Mannello, G.A.M. Tonti, and F. Canestrari The `never-ending story' of the influence of blood specimen collection methods affecting the concentration, the zymographic profile and the usefulness of matrix metalloproteinases and their tissue inhibitors in multiple sclerosis diagnosis/prognosis: a landmark for limiting the misuse of serum samples Multiple Sclerosis, June 1, 2007; 13(5): 687 - 690. [PDF]
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 A. Mazzoni, F. Mannello, F.R. Tay, G.A.M. Tonti, S. Papa, G. Mazzotti, R. Di Lenarda, D.H. Pashley, and L. Breschi Zymographic Analysis and Characterization of MMP-2 and -9 Forms in Human Sound Dentin J. Dent. Res., May 1, 2007; 86(5): 436 - 440. [Abstract] [Full Text] [PDF]
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E. Fainardi, M. Castellazzi, T. Bellini, and F. Dallocchio Matrix metalloproteinases (MMP): determination of different forms by different techniques may require different preanalytical strategies Multiple Sclerosis, May 1, 2007; 13(4): 561 - 562. [PDF]
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 C.-Y. Wu, M.-S. Wu, E.-P. Chiang, Y.-J. Chen, C.-J. Chen, N.-H. Chi, Y.-T. Shih, G.-H. Chen, and J.-T. Lin Plasma Matrix Metalloproteinase-9 Level Is Better than Serum Matrix Metalloproteinase-9 Level to Predict Gastric Cancer Evolution Clin. Cancer Res., April 1, 2007; 13(7): 2054 - 2060. [Abstract] [Full Text] [PDF]
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 N J Peake, H E Foster, K Khawaja, T E Cawston, and A D Rowan Assessment of the clinical significance of gelatinase activity in patients with juvenile idiopathic arthritis using quantitative protein substrate zymography Ann Rheum Dis, April 1, 2006; 65(4): 501 - 507. [Abstract] [Full Text] [PDF]
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 M. John and K. Jung Pre-analytical conditions for the assessment of circulating MMP-9 and TIMP-1: consideration of pitfalls Eur. Respir. J., August 1, 2005; 26(2): 364 - 365. [Full Text] [PDF]
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 M. John and K. Jung Consideration of Important Preanalytical Conditions for the Assessment of Circulating Matrix Metalloproteinase-9 Am. J. Respir. Crit. Care Med., July 15, 2005; 172(2): 254 - 254. [Full Text] [PDF]
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S. Zucker and J. Cao Measurement of Matrix Metalloproteinases in Serum of Patients with Melanoma: Snarled in Technical Pitfalls Clin. Cancer Res., July 15, 2005; 11(14): 5069 - 5070. [Full Text] [PDF]
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A. Meisser, M. Cohen, and P. Bischof Concentrations of Circulating Gelatinases (Matrix Metalloproteinase-2 and -9) Are Dependent on the Conditions of Blood Collection Clin. Chem., January 1, 2005; 51(1): 274 - 276. [Full Text] [PDF]
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F. Mannello, F. Luchetti, B. Canonico, E. Falcieri, and S. Papa Measurements, Zymographic Analysis, and Characterization of Matrix Metalloproteinase-2 and -9 in Healthy Human Umbilical Cord Blood Clin. Chem., September 1, 2004; 50(9): 1715 - 1717. [Full Text] [PDF]
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