PO-26 - Whole blood rotational thromboelastometry (ROTEM) to detect hypercoagulability in patients with myeloproliferative neoplasms (MPN).

INTRODUCTION: Essential Thrombocythemia (ET) and Polycythemia Vera (PV) are two MPNs characterized by a "clonal" overproduction of one or more blood cell lines, hypercoagulability, and an increased incidence of thrombosis. ROTEM is a point of care global coagulation assay performed in whole blood, able to evaluate platelets and fibrinogen contributions to the clotting process. Until now few studies evaluated the thromboelastometry profile of MPN patients. AIM: This study assess the feasibility of using ROTEM to characterize the prothrombotic state of MPN patients and to evaluate whether the thromboelastometry profile varies according to mutational status and/or treatment, and is influenced by hemocromocytometric parameters. MATERIALS AND METHODS: Venous blood samples were collected from 39 ET and 23PV patients upon informed consent. Analysis was performed using INTEM and EXTEM reagents, to evaluate the intrinsic and extrinsic pathway, respectively. Maximum clot firmness (MCF, [mm]), which reflects the maximum tensile strength of the thrombus, clotting formation time (CFT [sec]), namely the time that clot takes to increase from 2 to 20mm above baseline, and clotting time (CT [sec]), the time to clot initiation, were recorded. Nineteen healthy subjects acted as a control group. RESULTS: ROTEM analysis showed a hypercoagulable profile in MPN patients, who had shorter CFT and higher MCF compared to controls, both with EXTEM and INTEM reagents; no differences were observed in CT parameters. Platelet count was significantly higher in patients compared to controls (p<0.01). In patients, a strong statistically significant (p<0.01) correlation was found between platelet count, and MCF [r=0.650 (ET), r=0.601 (PV)] or CFT [r=-0.641 (ET), r=-0.558 (PV)]. Multivariate analysis, according to blood cell counts, showed that only platelet count was independently associated to ROTEM results. To correct for platelet differences, a ratio between MCF and the respective platelet value (rMCF) was created. Interestingly, rMCF was significantly lower in patients compared to controls (p<0.01), suggesting a weaker clot formation potential of patients' samples. Furthermore, rMCF was lower in ET compared to PV (p<0.05), and in calreticulin-positive subjects (p<0.05), while was higher in patients under cytoreductive therapy (Hydroxyurea) (p=ns). CONCLUSIONS: This study confirms, by the ROTEM evaluation, the occurrence of a hypercoagulable state in ET and PV patients. In addition, the ROTEM parameters are significantly influenced by the platelet count. Finally, MCF values corrected for platelet count reveal a lower platelet reactivity in MPN patients, confirming the hypothesis that platelet function is exhausted upon clotting activation. ACKNOWLEDGEMENT: Project funded by "AIRC-IG2013" grant Nr. 14505 from the "Italian Association for Cancer Research" (A.I.R.C.).

Storage method, drying processes and extraction procedures strongly affect the phenolic fraction of rosemary leaves: an HPLC/DAD/MS study.

The Rosmarinus officinalis L. is widely known for its numerous applications in the food field but also for the increasing interest in its pharmaceutical properties. Two groups of compounds are mainly responsible for the biological activities of the plant: the volatile fraction and the phenolic constituents. The latter group is mainly constituted by rosmarinic acid, by a flavonoidic fraction and by some diterpenoid compounds structurally derived from the carnosic acid. The aim of our work was to optimize the extractive and analytical procedure for the determination of all the phenolic constituents. Moreover the chemical stability of the main phenols, depending on the storage condition, the different drying procedures and the extraction solvent, have been evaluated. This method allowed to detect up to 29 different constituents at the same time in a relatively short time. The described procedure has the advantage to being able to detect and quantify several classes of compounds, among them numerous minor flavonoids, thus contributing to improving knowledge of the plant. The findings from this study have demonstrated that storing the raw fresh material in the freezer is not appropriate for rosemary, mainly due to the rapid disappearing of the rosmarinic acid during the freezing/thawing process. Regarding the flavonoidic fraction, consistent decrements, were highlighted in the dried samples at room temperature if compared with the fresh leaf. Rosmarinic acid, appeared very sensitive also to mild drying processes. The total diterpenoidic content undergoes to little changes when the leaves are freeze dried or frozen and limited losses are observed working on dried leaves at room temperature. Nevertheless it can be taken in account that this fraction is very sensitive to the water presence during the extraction that favors the conversion of carnosic acid toward it oxidized form carnosol. From our findings, it appear evident that when evaluating the phenolic content in rosemary leaves, several factors, mainly the type of storage, the drying process and the extraction methods, should be carefully taken into account because they can induce partial losses of the antioxidant components.

Anthocyanins and flavan-3-ols from grapes and wines of Vitis vinifera cv. Cesanese d'Affile.

The objective of the present study was to evaluate the amount of some potential health-promoting phenols in the grape of Vitis vinifera cv. Cesanese d'Affile and in wines made from these grapes. The analyses were performed using HPLC/DAD/MS. The accumulation of anthocyanins in the skin and flavan-3-ols in the seed was determined at different stages of ripening of the grape (i.e. green, veraison, middle stage of ripening, and complete ripening). Thirteen anthocyanins were identified in the skin at all stages of ripening, except the green stage. With regard to flavan-3-ols, (+)-catechin, (-)-epicatechin, and (-)-epicatechin gallate were detected in all of the seed samples. The highest (+)-catechin content was found in the seeds of the green grape (2 mg g(-1) DW), whereas in the seeds from the completely ripe grape the content was more than ten times lower. The highest catechin content in the seed was correlated with the lowest anthocyanin content in the skin. The wines produced in the years 2004 and 2005 showed, at wavelengths of 520 and 280 nm, almost identical quali-quantitative chromatographic profiles, with high concentrations of anthocyanin 3-O-glucosides, low concentrations of acylated anthocyanins, and trace amounts of (+)-catechin and (-)-epicatechin.

Commercial and laboratory extracts from artichoke leaves: estimation of caffeoyl esters and flavonoidic compounds content.

Artichoke leaf extracts are widely used alone or in association with other herbs for embittering alcoholic and soft drinks and to prepare herbal teas or herbal medicinal products. Despite this wide diffusion, the European Pharmacopoeia does not report an official method for the determination of the active principles of artichoke leaf extracts. This work reports a quali-quantitative determination by HPLC/DAD and HPLC/MS techniques of both cynnamic acids and flavonoids present in some artichoke leaf commercial extracts (Com) compared with two different laboratory extracts (Lab). Most of the commercial extracts showed a similar quali-quantitative pattern with a single exception having five-six times higher value. The quantitative data from the Italian Pharmacopoeia(IP) official method does not evaluate the flavonoidic fraction and showed an overestimation of the caffeoyl esters with respect to the HPLC/DAD results. The proposed HPLC/DAD method was able to completely characterize and quantify this matrix and represents a contribution to better quality control of these herbal extracts.

Polyphenolic content in olive oil waste waters and related olive samples.

The production of olive oil yields a considerable amount of waste water, which is a powerful pollutant and is currently discarded. Polyphenols and other natural antioxidants, extracted from olives during oil extraction process, partially end up in the waste waters. Experimental and commercial olive oil waste waters from four Mediterranean countries were analyzed for a possible recovering of these biologically interesting constituents. Identification and quantitation of the main polyphenols were carried out by applying HPLC-DAD and HPLC-MS methods. Representative samples of ripe olives were also analyzed at the same time to correlate, if possible, their polyphenolic profiles with those of the corresponding olive oil waste waters. The results demonstrate that Italian commercial olive oil waste waters were the richest in total polyphenolic compounds with amounts between 150 and 400 mg/100 mL of waste waters. These raw, as yet unused, matrices could represent an interesting and alternative source of biologically active polyphenols.

Stabilisation of natural anthocyanins by micellar systems.

In this paper, we discuss the influence of different micellar systems on the degradation of natural anthocyans, either glycosides and aglycones, at pH values ranging from 2.8 to 6.0. The interaction of anthocyanins, in suitable dispersed systems such as negative micelles of sodium dodecylsulphate (SDS), consistently increased their chemical stability in aqueous solutions. The results of these experiments point out how both the number of available negative charges and the presence of an organised distribution of the negative charges on the micellar surface appear to be necessary conditions to achieve the anthocyanins' stability and colour retention. The sodium dodecylbenzensulphonate (SDBS), containing an aromatic ring near the negative surface of the micelle, seems to increase the rate of decomposition. Preliminary findings of circular dicroism (CD) investigation allowed us to hypothesise that these pigments undergo an intermolecular self-association process induced by the SDS micelles and this phenomenon presumably contribute to increase stability.