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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@uniurb.it
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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
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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