Reply:
Authors: Robert J. Fontana
Journal: Hepatology (2008)

We thank Drs. Mannello and Jung for their comments regarding the influence of blood sample collection methods on the quantification of matrix metalloproteinase (MMP) and tissue inhibitor of matrix metalloproteinase (TIMP) levels. The activity of these endopeptidases is regulated at the tissue level via various soluble cytokines and interaction with platelets and white blood cells during inflammation and repair. TIMPs form one-to-one noncovalent complexes with MMPs and are widely synthesized by many cells and tissues. Recently, the blood level of various MMPs and TIMPs have been proposed as useful biomarkers for hepatic fibrosis severity. In particular, the tissue and serum levels of TIMP-1 strongly correlate with liver fibrosis severity in hepatitis C virus (HCV) and other liver diseases.1, 2 However, the available TIMP-1 assays recognize both free TIMP-1 and that complexed with circulating MMPs in the blood. Recently, the importance of blood sample handling on the quantification of various MMP and TIMP levels in patients with cancer and atherosclerosis have been reported. For example, MMP-2 and TIMP-2 levels do not substantially differ in plasma versus serum samples whereas MMP-1, MMP-3, and MMP-9 levels are substantially lower in plasma compared to serum.3, 4 The consistently higher levels of TIMP-1 in serum versus plasma may, in part, be due to the release of TIMP-1 from circulating blood elements during the processing and handling of serum samples, as suggested by Manello and Jung in healthy volunteers. Serum samples in our study were prospectively collected and processed according to a standard, written protocol at the four participating Hepatitis C Antiviral Long-Term Treatment Against Cirrhosis trial (HALT-C) sites. Frozen sera were then assayed for TIMP-1 using a commercial enzyme-linked immunosorbent assay kit. Statistically and clinically significant associations between the serum TIMP-1 levels and severity of hepatic fibrosis by both light microscopy and computerized morphometry were noted.5 Two serum fibrosis marker panels have also demonstrated significant associations between serum TIMP-1 levels and liver fibrosis severity in patients with HCV, fatty liver, and other liver diseases.6-8 Therefore, serum was selected as the analyte of choice when the HALT-C study was initiated in 2000, based on the information available at that time. Currently, we are unable to retrospectively determine if serum TIMP-1 levels are consistently higher than plasma TIMP-1 levels in the HALT-C patients and whether any potential differences would alter the utility of this marker in predicting liver fibrosis severity. Going forward, it will be worthwhile to compare the results of serum versus plasma TIMP-1 levels in future studies because the disease state itself may influence assay results in addition to specimen processing. Finally, the utility of TIMP-1 and other proposed noninvasive markers will require validation in longitudinal studies of liver disease progression and clinical outcomes. Robert J. Fontana M.D.*, * Department of Internal Medicine, University of Michigan Medical Center, Ann Arbor, MI.