Heparin-induced lipoprotein precipitation apheresis in dyslipidemic patients: A multiparametric assessment.

Low-density lipoprotein (LDL) apheresis (LA) selectively eliminates lipoproteins containing apolipoprotein B 100 (ApoB100) on patients affected by severe dyslipidemia. In addition to lowering lipids, LA is thought to exert pleiotropic effects altering a number of other compounds associated with atherosclerosis, such as pro- and anti-inflammatory cytokines or pro-thrombotic factors. More knowledge needs to be gathered on the effects of LA, and particularly on its ability to modify blood components other than lipids. We performed a multiparametric assessment of the inflammatory, metabolic and proteomic profile changes after Heparin-induced lipoprotein precipitation (H.E.L.P.) apheresis on serum samples from nine dyslipidemic patients evaluating cholesterol and lipoproteins, plasma viscosity and density, metabolites, cytokines, PCSK9 levels and other proteins selectively removed after the treatment. Our results show that H.E.L.P. apheresis is effective in lowering lipoprotein and PCSK9 levels. Although not significantly, complement and inflammation-related proteins are also affected, indicating a possible transient epiphenomenon induced by the extracorporeal procedure.

SANIST: a rapid mass spectrometric SACI/ESI data acquisition and elaboration platform for verifying potential candidate biomarkers.

RATIONALE: Surface-Activated Chemical Ionization/Electrospray Ionization mass spectrometry (SACI/ESI-MS) is a technique with high sensitivity and low noise that allows accurate biomarker discovery studies. We developed a dedicated SACI/ESI software, named SANIST, for both biomarker fingerprint data acquisition and as a diagnostic tool, using prostate cancer (PCa) as the disease of interest. METHODS: Liquid chromatography (LC)/SACI/ESI-MS technology was employed to detect a potential biomarker panel for PCa disease prediction. Serum from patients with histologically confirmed or negative prostate biopsies for PCa was employed. The biomarker data (m/z or Thompson value, retention time and extraction mass chromatogram peak area) were stored in an ascii database. SANIST software allowed identification of potential biomarkers. A Bayesian scoring algorithm developed in house allowed sample separation based on comparison with samples in the database. RESULTS: Biomarker candidates from the carnitine family were detected at significantly lower levels in patients showing histologically confirmed PCa. Using these biomarkers, the SANIST scoring algorithm allowed separation of patients with PCa from biopsy negative subjects with high accuracy and sensitivity. CONCLUSIONS: SANIST was able to rapidly identify and perform a preliminary evaluation of the potential diagnostic efficiency of potential biomarkers for PCa.

Reduction of EpCAM-Positive Cells from a Cell Salvage Product Is Achieved by Leucocyte Depletion Filters Alone.

Intraoperative cell salvage reduces the need for allogeneic blood transfusion in complex cancer surgery, but concerns about the possibility of it re-infusing cancer cells have hindered its application in oncology. We monitored the presence of cancer cells on patient-salvaged blood by means of flow cytometry; next, we simulated cell salvage, followed by leucodepletion and irradiation on blood contaminated with a known amount of EpCAM-expressing cancer cells, assessing also residual cancer cell proliferation as well as the quality of salvaged red blood cell concentrates (RBCs). We observed a significant reduction of EpCAM-positive cells in both cancer patients and contaminated blood, which was comparable to the negative control after leucodepletion. The washing, leucodepletion and leucodepletion plus irradiation steps of cell salvage were shown to preserve the quality of RBCs in terms of haemolysis, membrane integrity and osmotic resistance. Finally, cancer cells isolated from salvaged blood lose their ability to proliferate. Our results confirm that cell salvage does not concentrate proliferating cancer cells, and that leucodepletion allows for the reduction of residual nucleated cells, making irradiation unnecessary. Our study gathers pieces of evidence on the feasibility of this procedure in complex cancer surgery. Nevertheless, it highlights the necessity of finding a definitive consensus through prospective trials.

SPECT radiopharmaceuticals for dementia.

Over the last decade the interest towards functional neuroimaging has gradually increased, especially in the field of neurodegenerative diseases. At present, diagnosis of dementia is mostly clinical. Numerous modalities of neuroimaging are today available, each of them allowing a different aspect of neurodegeneration to be investigated. Although during the last period many have predicted a forthcoming disappearance of SPECT imaging in favour of the PET imaging, many new radiotracers SPECT, dual-SPECT tracers techniques and receptor targeting designed radiopharmaceuticals are currently at study. Besides, last decade has also assisted to the development of new SPECT imaging systems, most of them integrated with other imaging modalities (MRI, CT, ultrasound techniques), granting improved imaging capabilities. All these improved conditions, especially appealing for the neuroimaging, together with the new radiopharmaceuticals in development may renovate the interest for SPECT clinical applications.

Brief report on the use of radiolabeled somatostatin analogs for the diagnosis and treatment of metastatic small-cell lung cancer patients.

INTRODUCTION: The demonstration of type 2 somatostatin receptors (SSTRs) in small-cell lung cancer (SCLC) represents the rationale for the use of positron emission tomography/computed tomography (PET/CT) to determine SSTR expression, and select patients suitable for peptide radioreceptor radionuclide therapy (PRRT) in extensive-disease stage (ED) SCLC. METHODS: We evaluated 24 ED-SCLC patients with radiolabeled SST-analog PET/CT. Lesions at PET/CT scan were semiquantitatively scored (from 0 to 3+) and compared with contrast-enhanced CT findings. Patients scored as 3+ were admitted to PRRT after dosimetric evaluation. Average injected activity/cycle was 2.6 GBq (yttrium-PRRT) or 6.0 GBq (lutetium-PRRT). PRRT efficacy was clinically and radiologically assessed. RESULTS: PET/CT was negative in four of 24 patients, whereas in the remaining 20 cases uptake was scored as 1+ in seven of 20, 2+ in one of 20, and 3+ in 12 of 20. Primary tumor lesions showed uptake in 16 of 24 patients. Uptake in metastatic lesions was observed in four of four adrenals, two of five brain, 12 of 16 bone, three of eight liver, and 17 of 20 lymph node lesions. Of the 12 patients eligible for PRRT, 11 were eventually treated and four of 11 patients received multiple PRRT administrations. Dosimetry resulted in a BED for kidney of 7.5 Gy (range, 4-21); bone marrow provisional dosage was 0.43 Gy (range, 0.1-1.7). Hematological PRRT toxicity occurred in three of 11 patients. No clinical or objective responses were observed with disease progression occurring approximately 48 days (range, 9-32) after PRRT. CONCLUSION: Radiolabeled SST-analog PET/CT demonstrated enhanced SSTR expression in 50% of cases. Nevertheless, PRRT in ED-SCLC was ineffective, suggesting the need to anticipate or combine PRRT in a multimodality approach.

Semiautomated labelling and fractionation of yttrium-90 and lutetium-177 somatostatin analogues using disposable syringes and vials.

OBJECTIVES: The treatment of tumours expressing somatostatin receptors with yttrium-90 (90Y)-labelled and lutetium-177 (177Lu)-labelled somatostatin analogues is one of the most interesting therapeutic approaches adopted in nuclear medicine in recent years. However, the process of synthesis and fractionation of these radiopharmaceuticals is still mainly carried out manually despite the high radiation exposure to the operators and the need to comply with good manufacturing practices. In this study a semiautomatic synthesizer [automatic dose dispenser (ADD-2)] using only disposable syringes and vials has been presented. MATERIALS AND METHODS: Small-scale syntheses (185-555 MBq) of 90Y/177Lu-DOTATATE were performed by adding the appropriate amount of peptide to a 90Y/177Lu chloride solution (n=10). The radionuclide/peptide molar ratio was 1 : 17 and 1 : 2 for 90Y and 177Lu, respectively. The solutions were buffered to 4.6 pH by ascorbate buffer and heated at 90°C for 30 min. Radiochemical purity was assessed by two independent radio-thin-layer chromatography systems. The solutions were fractioned to mimic the preparation of patient doses. RESULTS: All synthesis and fractionation steps were performed using ADD-2. The radiochemical yield was 92 ± 3% for 90Y and 97 ± 1% for 177Lu labelling. Radiochemical purity was more than 99.5%. The accuracy and reproducibility of the instrument in transferring and fractionating radioactive solutions were high (maximal error ≈ 5%). CONCLUSION: ADD-2 appears suitable for use in clinical preparations of 90Y/177Lu-DOTATATE with therapeutic amounts of precursors (20-30 GBq). The operator's exposure to radiation by using ADD-2 in comparison with manual preparations is under investigation.

Radiosynthesis of 68Ga-labelled DOTA-biocytin (68Ga-r-BHD) and assessment of its pharmaceutical quality for clinical use.

OBJECTIVES: Biocytin analogues labelled with indium-111, yttrium-90 and lutetium-177 have shown their effectiveness in the imaging of infections/inflammation in patients with osteomyelitis and function as efficient tools in pretargeted antibody-guided radioimmunotherapy. In this study, the labelling of a biocytin analogue coupled with DOTA (1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid), namely, r-BHD, with gallium-68 (68Ga) was optimized, and the quality and stability of the preparations were assessed for clinical use. MATERIALS AND METHODS: Synthesis of 68Ga-r-BHD was carried out by heating a fraction of the 68Ge/68Ga eluate in a reactor containing the biocytin analogue with the appropriate buffer. The influence of the precursor amount (from 2.5 to 140 nmol), the pH of the reaction (from 2 to 5.5) and the buffer species (1.5 mol/l sodium acetate, 1.5 mol/l sodium formate, 4.5 mol/l HEPES) on radiochemical yield and radiochemical purity was assessed. Studies on stability and binding to avidin (Av) were also conducted in different media. RESULTS: Under the best labelling condition (56 nmol of precursor, 3.8 pH, sodium formate buffer) synthesis of 68Ga-r-BHD resulted in a yield of 64 ± 3% (not decay corrected). Radiochemical purity was around 95% because a 68Ga-coordinated sulfoxide form of the ligand was detected as a by-product of the reaction (68Ga-r-SBHD). The by-product was identified and characterized by liquid chromatography-electrospray ionization tandem mass spectrometry. At the natural 1 : 4 Av/68Ga-r-BHD molar ratio, affinity results were 62 ± 2 and 80 ± 2% in saline and human serum, respectively. Stability of 68Ga-r-BHD and of the radiotracer/Av complex remains almost constant over 180 min. 68Ga-r-BHD appears to be a good candidate for clinical applications.

Influence of cations on the complexation yield of DOTATATE with yttrium and lutetium: a perspective study for enhancing the 90Y and 177Lu labeling conditions.

The DOTA macrocyclic ligand can form stable complexes with many cations besides yttrium and lutetium. For this reason, the presence of competing cationic metals in yttrium-90 and lutetium-177 chloride solutions can dramatically influence the radiolabeling yield. The aim of this study was to evaluate the coordination yield of yttrium- and lutetium-DOTATATE complexes when the reaction is performed in the presence of varying amounts of competing cationic impurities. In the first set of experiments, the preparation of the samples was performed by using natural yttrium and lutetium (20.4 nmol). The molar ratio between DOTATATE and these metals was 1 to 1. Metal competitors (Pb(2+), Zn(2+), Cu(2+), Fe(3+), Al(3+), Ni(2+), Co(2+), Cr(3+)) were added separately to obtain samples with varying molar ratio with respect to yttrium or lutetium (0.1, 0.5, 1, 2 and 10). The final solutions were analyzed through ultra high-performance liquid chromatography with an UV detector. In the second set of experiments, an amount of (90)Y or (177)Lu chloride (6 MBq corresponding to 3.3 and 45 pmol, respectively) was added to the samples, and a radio-thin layer chromatography analysis was carried out. The coordination of Y(3+) and Lu(3+) was dramatically influenced by low levels of Zn(2+), Cu(2+) and Co(2+). Pb(2+) and Ni(2+) were also shown to be strong competitors at higher concentrations. Fe(3+) was expected to be a strong competitor, but the effect on the incorporation was only partly dependent on its concentration. Al(3+) and Cr(3+) did not compete with Y(3+) and Lu(3+) in the formation of DOTATATE complexes.

Efficient automated one-step synthesis of 2-[18F]fluoroethylcholine for clinical imaging: optimized reaction conditions and improved quality controls of different synthetic approaches.

UNLABELLED: [(18)F]-labelled choline analogues, such as 2-[(18)F]fluoroethylcholine ((18)FECH), have suggested to be a new class of choline derivatives highly useful for the imaging of prostate and brain tumours. In fact, tumour cells with enhanced proliferation rate usually exhibit an improved choline uptake due to the increased membrane phospholipids biosynthesis. The aim of this study was the development of a high yielding synthesis of (18)FECH. The possibility of shortening the synthesis time by reacting all the reagents in a convenient and rapid one-step reaction was specially considered. METHODS: (18)FECH was synthesized by reacting [(18)F]fluoride with 1,2-bis(tosyloxy)ethane and N,N-dimethylaminoethanol. The synthesis was carried out using both a one- and a two-step reaction in order to compare the two procedures. The effects on the radiochemical yield and purity by using different [(18)F]fluoride phase transfer catalysts, reagents amounts and purification methods were assessed. Quality controls on the final products were performed by means of radio-thin-layer chromatography, gas chromatography and high-performance liquid chromatography equipped with conductimetric, ultraviolet and radiometric detectors. RESULTS: In the optimized experimental conditions, (18)FECH was synthesized with a radiochemical yield of 43+/-3% and 48+/-1% (not corrected for decay) when the two-step or the one-step approach were used, respectively. The radiochemical purity was higher than 99% regardless of the different synthetic pathways or purification methods adopted. The main chemical impurity was due to N,N-dimethylmorpholinium. The identity of this impurity in (18)FECH preparations was not previously reported. CONCLUSION: An improved two-step and an innovative one-step reaction for synthesizing (18)FECH in a high yield were reported. The adaptation of a multistep synthesis to a single step process, opens further possibilities for simpler and more reliable automations.