In vitro Research Concerning Effect of Clopidogrel Alone and on Combination with Aspirin and Dypiridamoleon Sedimentation of Erythrocytes.

Based on extended theory of Derjaguin, Landau and Overbeeck (xDLVO) concerning aggregation of colloids and biological cells it was hypothesized that platelet antiaggregant agents have to reduce the aggregation of erythrocytes also. Applying Einstein-Stokes theory of sedimentation of spheres in viscous media it was concluded that sedimentation of erythrocytes is in fact sedimentation of aggregates of the approximately same size. Consequently, an expected outcome was that addition of antiaggregants in vitro to blood samples from patients with rheumatic or cardiovascular diseases will be the decrease of erythrocytes sedimentation. Starting from usual practice of dual antiaggregant therapy (aspirin and clopidogrel) effects of clopidogrel were compared with effects of clopidogrel plus small concentrations of aspirine and dipyridamole (smaller that their concentrations in plasma after in vivo administration) in order to put in evidence a possible synergic effect at platelet membrane level. Whole blood (0.8ml) was collected on 0.1ml 1% EDTA and then was added 25 or 50µl normal saline solution of clopidogrel or of the combination acetylosalycilic acid, clopidogrel and dypyridamole. The final concentrations were 1, 2 and µg/ml, of the same order as cumulated concentration of clopidogrel and its metabolites in clinical pharmacokinetics. Experiments were performed on a number of 40 human blood samples obtained from 2 groups of 20 patients. Sedimentation of erythrocytes was recorded using a camera and captured data were stored on a computer. Global analysis evidenced that in presence of antiaggregants the clusters of the sedimentation curves shifted down and into right, indicating a decrease and delay of sedimentation. Initial slopes and extent of sedimentation decreased linearly on clopidogrel concentration within the 1-3µg/ml range. For comparison of mean curves corresponding to different clopidogrel concentrations it was applied a metric from biopharmacy: areas under plasma concentrations curves (AUC) of drugs. The areas under average sedimentation curves decreased linearly at clopidogrel concentration within the 1-3µg/ml range. The same experiments were performed and similar results were obtained with the triple antiaggregant combination (clopidogrel, acetylosalycilic acid and dipyridamole). Apparently, a synergism between the tested antiaggregants appeared at studied concentration but the number of data was not sufficient to prove the statistical significance of the difference between clopidogrel alone and in triple combination.

In vitro Research of the Concentration Dependence of Effect of Adrenaline on Platelets Aggregation.

PURPOSE: Paper intended to present experimental evidences that adrenaline has a direct effect of inducing platelets aggregation in the concentration range 1-8µM. MATERIAL/METHODS: Platelet rich plasma from patients of Colentina Clinical Hospital, following an informed consent. The platelet rich plasma (PRP) was prepared by centrifuging the anticoagulated sample at 200 G for 10 minutes. Aggregation was evaluated by optical aggregometry, classical method of Born, using Helena PACKS-4 Aggregometer. RESULTS: The curves transmission light-time followed the structure: a lag-time, a first phase aggregation, more or less linear, defined by a "Slope 1", a second wave of aggregation defined by "slope 2" and a "saturation" phase. Slope 1 increases with the concentration of adrenaline. The second slopes of the aggregation curves, maximum aggregation and areas under curves depended linear on adrenaline concentration. CONCLUSIONS: Adrenaline, in concentrations in the 1-8µM, induce aggregation of human platelets from platelet rich plasma. Linear regression models for slope and area were practically identical suggesting a rather unique than biphasic mechanism of action of adrenaline during the time course of aggregation.

Application of Biopharmaceutical and Clinical Metrics in Analyzing Modification of Platelets Aggregation Curves by Calcium Ion.

In context of Evidence Based Medicine concept, Good Clinical Practice rules specify that "data generated should be reliable and robust".Reliability and robustness are further translated in requirements concerning statistical and clinical significance of results. Paper presents main aspectsconnected with comparison of evolutions of endpoints as function of different parameters like time, administered dose, proportion of active components etc., leading to problems of comparison of curves, with direct application to comparison of platelets aggregation curves in presence of different concentrations of ionic calcium. Theoretical part presents comparison of curves in biopharmacyusing f2 metric and area under curve metric, and comparison of survival curves in clinical studies.Platelet aggregation test was performed using Bornturbidimetric light transmission method using Helena PACKS-4 Aggregometer. Blood samples were collected from patients in internal medicine ward of Colentina Clinical Hospital. Platelet rich plasma (PRP) was obtained by centrifugation at 200G. Washed platelets where extracted by centrifugation of PRP at 2700G. The supernatant was replaced with sodium chloride 0.9%. Platelets aggregation was induced by adding different concentrations of calcium gluconate into cuvettes which contained washed platelets. After digitalization, curves were compared using similarity factor f2and areas under curves. Paper puts in evidence that both type of comparison, after mathematical and statistical evaluation, have to define a clinical threshold for clinical significance. In case of f2, in dissolution studies the threshold is 10%, in case of bioequivalence based on area under curves threshold is 20%. Establishment of the threshold for significant clinical difference in comparison of aggregation curves is not only a problem of statistics.Graphical representation of data suggested significant differences between curves obtained with different concentrations of calcium ion. Application of both f2 method and log-rank test let to conclusion that differences were statistical significant. Representation of aria under curves as function of calcium concentration put in evidence an approximate linear dependence. In spite of apparently objective character of mathematical approach, the problem of comparison of aggregation curves remains practically unsolved since we do not know the threshold between clinical significant and non-significant results.

[Catheter for continuous interpectoral block and postoperative pain relief in breast surgery]. / Bloc interpectoral avec mise en place d'un cathéter pour analgésie postopératoire après chirurgie mammaire.

We describe the case of four patients who had undergone breast surgery. The post-operative analgesia consisted in levobupivacaine administered through a catheter placed between the pectoralis minor and the pectoralis major using ultrasound guided technique. The quality of the analgesia was excellent quality. No side-effects were encountered.