HMGB1 expression in leukocytes as a biomarker of cellular damage induced by [99mTc]Tc-HMPAO-labelling procedure: a quality control study

Purpose. Autologous White Blood Cells (WBC) scintigraphy is based on a multi-step sequence of cell separation and radiolabelling. Besides in vivo imaging quality control, no molecular tool is available to evaluate WBC damage secondary to cell manipulation. High Mobility Group Box 1 (HMGB1) is a protein of the alarmins family, secreted by innate immune cells and released from the nucleus of damaged cells following different types of injury. Aim of this study was to evaluate HMGB1 levels in WBC cytosolic extracts (CE) before and after [99mTc]Tc-HMPAO labelling procedure, as a biomarker of induced WBC damage. Procedures. Patients with suspect of prosthetic joint infection were prospectively enrolled. HMGB1 levels were evaluated by immunoblotting analysis in plasma (t0), and in WBC-CE before (t1) and after (t2) [99mTc]Tc-HMPAO labelling. Blood samples from healthy subjects were evaluated under the same procedure. Results. Twenty consecutive patients referred for WBC scintigraphy and ten controls were enrolled. HMGB1 levels were significantly upregulated both in plasma (t0) and in circulating WBC-CE (t1) from patients compared to controls (p<0.0001). Otherwise, WBC-CE from [[99mTc]Tc-HMPAO-labelled leukocyte concentrate (t2) did not show significant changes in HMGB1 levels compared to the cold leukocyte sample (t1). Conclusions. The evaluation of HMGB1 levels in WBC-CE from each subject after radiolabelling with [99mTc]Tc-HMPAO did not show significant changes compared to the cold cellular sample. These results further prove the reliability of [99mTc]Tc-HMPAO leukocyte radiolabelling procedure in terms of cell viability and suggest that the monitoring of this alarmin may represent a specific tool to evaluate a secondary damage of WBC induced by radiolabelling procedure. In addition, significant upregulation of HMGB1 levels was found in WBC-CE and in plasma from patients with suspect of PJI - compared to healthy donors - reasonably related to their underlying inflammatory/infective condition.