Development and preliminary use of CDT analysis on dried blood spot (DBS) in forensic and administrative contexts

Abstract

BACKGROUND-AIM Carbohydrate Deficient Transferrin (CDT) defines two minor glycoforms of transferrin (asialo and disialo-tansferrin), characterized by a reduced glycosylation degree, whose serum concentration increases after chronic sustained alcohol intake (60-80 g per day for at least 10 days). The use of finger-prick and related dried blood spots (fpDBS) is an innovative tool for blood sample collection in clinical and forensic toxicology. The aim of this work was to develop a screening method for CDT analysis based on the use of fpDBS coupled with capillary electrophoresis. METHODS Capillary blood drops collected by finger-prick were placed on DBS cards and left to air dry. Each dried fpDBS disc was sliced and suspended in acid solution. After centrifugation the sample pH was adjusted by 120 mmol HCl to pH 3-4. After overnight incubation the sample pH was neutralized and an iron rich solution was added. The resulting sample was analyzed by a validated CE method. The CDT level was expressed as %CDT (%ratio of disialo-Tf on total transferrin). The blood samples were obtained from volunteers of the forensic toxicology laboratory and from subjects submitted to blood testing for mandatory toxicological investigations. The DBS were analyzed in parallel with the sera of each investigated subject, using HPLC and CE techniques. The %CDT cut-offs used for the study were 1.80% and 1.90% for CE and HPLC, respectively. RESULTS The observed fpDBS transferrin glycoform CE patterns were comparable with serum CE CDT patterns. Moreover, a statistical correlation was demonstrated of fpDBS CDT percentage levels with both HPLC and CE % CDT (p< 0.01). This correlation was confirmed also by Passing-Bablok tests and Bland Altman test. The cut off proposed for this %CDT screening method was 1.6% demonstrating a sensitivity and specificity of about 75% and 90%, respectively. These data were calculated comparing %CDT by fpDBS CE vs serum HPLC, the latter considered the reference method. CONCLUSIONS The results of the study, even if preliminary, showed that fpDBS procedure coupled with CE for CDT analysis could express a simplified and inexpensive tool designed for use in population screening