Detecting Autologous Blood Transfusion in Doping Control: Biomarkers of Blood Aging and Storage Measured by Flow Cytofluorimetry.

BACKGROUND: Blood transfusions are banned by the World Anti-Doping Agency as a form of "blood doping". A method of detection of homologous blood transfusion (HBT) has been implemented by the accredited anti-doping laboratories worldwide; however, no internationally recognized method has been finalized so far for the direct detection of autologous blood transfusions, which can at present be revealed only by targeted longitudinal profiling of key blood parameters. METHODS: The present article reports the results of an investigation aimed to pre-select potential biomarkers of blood aging and storage that can be measured to identify the presence in the sample of reinfused blood. Microparticles from platelets and erythrocytes, erythrocytes size and density, annexin V (as a marker of phosphatidylserine externalization), and the membrane surface antigens CD 55 and CD 59, were specifically considered as potential biomarkers and measured by flow cytofluorimetric techniques. RESULTS AND CONCLUSION: Our results indicate that the parameters more strongly affected by the ex vivo storage of whole blood are erythrocytes size and density, annexin V and microparticles. Although the real diagnostic value of the proposed biomarkers shall obviously be confirmed by further studies carried out on blood samples collected after an actual autologous blood transfusion, these results appear very encouraging towards the development of a direct method for detecting autologous blood transfusion in sport doping.

Wnt signaling as target for the treatment of osteoarthritis.

Osteoarthritis is a severe and common rheumatic and skeletal disease for which currently no specific drugs are available. The Wnt signaling pathway modulates key biological processes in development, growth, homeostasis, and disease, particularly in the joints and bone. Excessive activation of the Wnt signaling pathway in the articular cartilage has been clearly associated with the onset and severity of osteoarthritis. Hence, targeting Wnt signaling may be an excellent approach to develop specific drugs useful for the treatment of osteoarthritis. In this article, we review the biology of Wnt signaling in the context of osteoarthritis; we also analyze the gradual improvement of our molecular understanding of Wnts in the joint and oversee current progress toward the development of Wnt inhibition as therapy for osteoarthritis. At least one Wnt inhibitor is currently going forward in the clinical evaluation process, potentially marking the beginning of a new era in the management of osteoarthritis.

Application of DNA-based forensic analysis for the detection of homologous transfusion of whole blood and of red blood cell concentrates in doping control.

In this work we present the application of a method for the identification of homologous blood transfusions using forensic genetic techniques based on DNA typing. Ex vivo mixtures of human blood samples - either whole blood or red blood cell concentrates - simulating homologous blood transfusions at different percentages of the donor were typed for a panel of 16 highly variable DNA short tandem repeats (STR). Tested samples included also mixtures, which gave false-negative results if assayed by the reference flow cytofluorimetric method, which is based on the recognition of target antigens located on the membrane of the red blood cell. The recognition of triplets and quadruplets at various loci gave information of the presence of cells belonging to different individuals, as it is the case for homologous blood transfusions. Specificity and sensitivity of the method were assessed in the validation study. The method proved to be unequivocally specific since it was able to recognize all single profiles of each individual, clearly discriminating them from mixtures. Sensitivity resulted as a consequence of the percentage of the donor aliquot in the total volume of the mixture. Although the source of DNA in a blood sample is represented only by nucleated white blood cells, the same procedure resulted effective also in detecting mixtures of red blood cell concentrates (RBCC) from leukodepletion procedure: DNA of the donor from the residual white blood cells resulted still detectable, even if with an expected loss of sensitivity. The proposed approach may contribute to reduce the risk of false-negative results, which may occur using the reference cytofluorimetric method.

Rapid prototyping of chitosan-coated alginate scaffolds through the use of a 3D fiber deposition technique.

Three dimensional, periodic scaffolds of chitosan-coated alginate are fabricated in a layer-by-layer fashion by rapid prototyping. A novel dispensing system based on two coaxial needles delivers simultaneously alginate and calcium chloride solutions permitting the direct deposition of alginate fibers according to any designed pattern. Coating of the alginate fiber with chitosan and subsequent cross-linking with EDC and genipin assured the endurance of the scaffold in the culture environment for a prolonged period of time. The cross-linking protocol adopted imparted to the scaffold a hierarchical chemical structure as evidenced by Confocal Laser Microscopy and FTIR spectroscopy. The core of the fibers making up the scaffold is represented by alginate chains cross-linked by ester bonds only, the periphery of the fiber is constituted by an inter-polyelectrolyte complex of alginate and chitosan cross-linked in all pair combinations. Fibers belonging to adjacent layers are glued together by the chitosan coating. Mechanical behavior of the scaffolds characterized by different layouts of deposition was determined revealing anisotropic properties. The biocompatibility and capability of the scaffolds to sustain hepatocyte (HepaRG) cultures were demonstrated. Typical hepatic functions such as albumin and urea secretion and induction of CYP3A4 enzyme activity following drug administration were excellent, thus proving the potential of these constructs in monitoring the liver specific function.

Investigation on the application of DNA forensic human identification techniques to detect homologous blood transfusions in doping control.

Homologous blood transfusion is an illicit practice used by athletes to improve the delivery of oxygen to tissues and, as such, it is banned in sports. The current method of detection is based on the flow cytofluorimetric phenotypic identification of red blood cells mismatch of minor blood group antigens between the donor and the recipient. The selectivity of this method to clearly identify transfused samples is related to the number of blood group antigens tested. Despite the fact that several different antigens are investigated, two individuals sharing the expression of the same minor blood group antigens pattern cannot be distinguished. We tested the possibility to use a different approach based on DNA forensic human identification techniques. Analysis of the DNA short tandem repeats (STRs) demonstrated its suitability in detecting mixed whole blood samples simulating homologous blood transfusion in 100% of the samples tested, ensuring the capability of clearly detecting mixed blood cell populations also on samples where the fraction of the minoritary population is as low as 2.5%.

Synthesis and characterization of a novel poly(vinyl alcohol) 3D platform for the evaluation of hepatocytes' response to drug administration.

Many whole cell-based assays in use today rely on flat, two-dimensional (2D) glass or plastic substrates that may not produce results characteristic of in vivo conditions. In this study, a three-dimensional (3D) cell-based assay scaffold was fabricated using a gas-in-foam templating technique. The scaffold was made of poly(vinyl alcohol), a water-soluble synthetic polymer with excellent film-forming, emulsifying, and biocompatible properties widely used in the biomedical field. The preliminary rheological studies on the solution of PVA and surfactant permitted us to disclose the significant physical parameters that influence the morphology of the ensuing materials. The scaffolds obtained were subjected to detailed analysis by light microscopy, Scanning Electron Microscopy (SEM), computed X-ray microtomography (µCT), infrared spectroscopy, and mechanical testing. Morphological investigations showed that the produced scaffolds are characterised by average void and interconnect diameters lying in the range of 200-300 and 30-150 µm, respectively, suitable for cell infiltration. Two different cross-linking procedures were adopted in order to modulate the mechanical properties of the PVA scaffolds. One made use of a bi-epoxide (PEGDGE), the other was based on glutaraldehyde (GA). The efficiency in terms of cross-linking density of the two procedures resulted in very different mechanical properties. Furthermore, in this article it is demonstrated how PVA foams can be processed into uniform, porous films suitable to be integrated with multi-well 2D culture plates in order to create a 3D analogue. The PEGDGE cross-linked scaffold was tested on C3A cells, a human hepatocyte cell line, representing an appropriate model for liver toxicity studies. Proliferation and cytotoxicity assays indicated good cell viability throughout the culture time, which was also confirmed by SEM analysis. Typical hepatic functions such as albumin and urea production and induction of Cyp3A4 enzyme activity following drug administration were satisfactory, thus proving the efficiency of this construct in maintaining specific liver functions.