Ten healthy adults (eight men, two women; age, 48 ± 17 years) with no blood biochemical abnormalities were included as a control group. Between November and December 2005, 60 patients (33 men, 27 women; age, 63 ± 12 years) with HCV-associated CLD, including 13 patients enrolled in the former study, were included in a study designed to assess the correlation between plasma stromal derived factor-1α (SDF-1α) concentrations

and CLD stage. Additionally, seven patients with HCV-associated LC, who had adequate liver function and underwent splenectomy between February 2005 and May 2007 (three men, four women; age, 55 ± 9 years), were enrolled in this study. Blood samples were collected preoperatively, at 1 week after surgery and at 1–3 months after surgery. Spleen samples were obtained from another three LC patients, who learn more underwent splenectomy within 1 year. Written informed consent

was obtained from all patients and healthy volunteers. The study protocol was approved by the Human Studies Subcommittee of Mie University Graduate School of Medicine (approval no. 287) and conformed to EGFR targets the ethical guidelines of the Declaration of Helsinki, 1975. Peripheral blood samples were drawn from patients with HCV-associated CLD and from healthy volunteers. Erythrocytes were lysed using ACK lysis buffer, and the remaining cells were washed twice with Ca2+/Mg2+-free phosphate-buffered saline (PBS; Gibco, Grand Island, NY, USA) and resuspended in PBS containing 0.1% de-ionized fraction V bovine serum albumin (BSA; Sigma-Aldrich, St. Louis, MO, USA). We used

the remaining cells as PB total nucleated cells (TNC) for flow cytometry. PB mononuclear cells (MNC) were prepared by density gradient centrifugation over Ficoll-Paque PLUS (GE Healthcare Bio-Sciences, Uppsala, Sweden) and were used for the CFU-C assay. Peripheral blood TNC were stained with fluorescein isothiocyanate (FITC)-conjugated antihuman CD34 (Becton Dickinson Immunocytometry Systems, San Jose, CA, USA) and phycoerythrin (PE)-conjugated antihuman CD90 (Becton Dickinson Pharmingen, San Diego, CA, USA) or PE-conjugated antihuman CD117 (Becton Dickinson Pharmingen). Relevant isotype-matched control antibodies were included in the staining to exclude non-specific binding. After adding 1 μg/mL of ioxilan propidium iodide (Sigma-Aldrich) to eliminate dead cells from the analysis, the cells were washed and resuspended in PBS containing 0.1% BSA. At least 200 000 events in live leukocyte populations were acquired by flow cytometry (FACSCalibur; BD Biosciences, San Jose, CA, USA) and were analyzed using CellQuest software (BD Biosciences). The number of CD34+ cells in living PB-TNC was determined using a CD34-FITC versus side-scatter dot plot. In some samples, PB-TNC were double stained with FITC-conjugated anti-human CD34 antibody and allophycocyanin-conjugated anti-human CD45 antibody (BioLegend, San Diego, CA, USA).