ALBERT

Description: Serotonergic mechanisms implied in both neuronal and platelet functions have been pointed out as a possible link between depressive disorders and cardiovascular risk. Recent studies suggest serotonin (5-HT) as a key element in the generation of a subpopulation of platelets with elevated procoagulant properties (coated-platelets). We have investigated the implication of serotonergic mechanisms in platelet and coagulation activation in samples from healthy donors or from patients suffering major depression. Furthermore, we evaluated the possible prothrombotic role of serotonergic mechanisms and the potential antithrombotic effect of selective serotonin reuptake inhibitors (SSRI) in vitro and compared results with those observed in a group of patients with major depression. Different experimental strategies were used to evaluate platelet function and the activation of coagulation system including standard aggregometry, flow cytometry, perfusion studies with circulating blood to evaluate platelet interaction and fibrin deposition on damaged vessels. Levels of tissue factor (TF) in whole blood and prothrombin fragment F1+2 in plasma samples were also determined. Aggregation studies did not reveal marked differences between control and patients with major depression. The presence of SSRI in plasma decreased up to 30% of the aggregation induced by ADP potentiated with 5-HT. Flow cytometry studies revealed that platelets from patients with major depression showed an enhanced expression of platelet antigens: FV, fibrinogen, CD63, GPIV, GPIb, as well as elevated exposure of anionic phospholipids (p

Description: Exposure of Tissue Factor (TF) to circulating blood and excessive thrombin generation are major contributors to acute thrombosis. Platelets also play a central role in the pathogenesis of occlusive events. Despite the importance of both components, convincing evidence of direct adherence of platelets onto TF immobilized on a surface is lacking. Here, we studied the interactions of platelets with different TF-rich substrata. Real-time perfusion techniques were applied to visualize thrombus formation and to evaluate the time necessary for platelets to both attach to the surface and to form thrombi. We used three different sources of TF as substrates:Thromborel S®, which is TF purified from human placenta (pTF) (at 0.4 ng/cm2);Innovin®, recombinant TF that has been relipidated (rTF) (at 0.4 ng/cm2); andTF expressed on the membranes of TF-transfected CHO cells (CHO-TF). As negative controls, we used equivalent surfaces sprayed with 0.5% bovine serum albumin or cultured sham-transfected CHO cells (CHO-C-), respectively. Platelet-rich plasma (PRP) from blood anticoagulated with low-molecular-weight heparin was perfused over these substrata at a shear rate of 300s−1 for 5 min. Images were captured with a digital camera at high resolution and streamed to a computer. Continuous real-time photographs were taken during the full 5 min. Single frames were captured at predetermined times and the mean area of the platelet aggregates at these times was measured. Every experiment was replicated at least 4 times. Results are expressed as mean ± S.E.M. Under these conditions, platelets bound and formed thrombi on all substrata containing TF, although the time to aggregate formation and aggregate area varied depending on the substratum. Platelet deposition was minimal on the BSA-coated surfaces or on CHO-C- cells. Initial platelet deposits on pTF were observed after 120 sec of perfusion. After 5 min, thrombi reached an area of 122±41 μm2. Relipidated rTF was less effective in supporting platelet deposition, requiring 135 seconds on average for the initiation of platelet aggregates. After 5 min, the average area of platelet aggregates was 34.3±9.6 μm2. Formation of platelet aggregates on CHO-TF cells was noticeable after 80 seconds. Aggregates formed at 5 min reached an area of 213±58 μm2. In conclusion, our studies demonstrate that platelets adhere to and aggregate on different TF substrata. The presence of other adhesive molecules in cell-derived TF preparations (pTF, CHO-TF) appears to accelerate the interaction of platelets with TF. These observations suggest that in addition to its role in blood coagulation, TF also has role in platelet adhesion by providing an adhesive surface at sites of vessel injury. SAF2003-05780, SGR2005-00952, FIS PI040887, FIS CP04-0011, NHLBI 5R01HL064796.

Description: Abstract 1347 Poster Board I-369 Introduction Sepsis is among the top 10 causes of death, but improvements in the diagnostic tests for detecting and monitoring sepsis and infection have been limited in the last years. Neutrophil CD64 expression increases rapidly in the presence of inflammation mediators and in response to infection and tissue damage. We have evaluated changes in the expression of neutrophil CD64 in infected patients in comparison with other markers of infection and sepsis. Methods Prospective analysis of 56 blood samples from patients from the intensive care unit at our institution was performed for neutrophil CD64 expression, C-reactive protein (CRP), automated absolute neuthophil count (ANC), and complete manual leucocyte formula including % of bands (BANDS), and % of metamyelocytes and myelocytes (IG). Neutrophil CD64 expression was measured by flow cytometry using a quantitative method (Leuko64TM, Trillium Diagnostics, LLC). Patients were categorized into 5 groups (CLINIC) based on the clinical history and the degree of a systemic inflammatory response, from 1 (no inflammation) to 5 (septic shock). Statistics were performed using linear regression, correlation coefficient, and Passing-Bablock (P-B) regression. Sensitivity (S), specificity (SP), efficiency (E), and positive and negative predictive values (PPV and NPV respectively) were analyzed for all the parameters measured. Results Our results showed a correlation with CLINIC of 0.417, 0.552, 0.268, and 0.136 for CD64, CRP, BANDS, and ANC, respectively. P-B regression was only good for CD64, with a slope of 1.03 (0.6-1.4). Percentages (%) of S, SP, E, PPV, and NPV for CD64 were of 81%, 72%, 71%, 46% and 92%, respectively for groups 4 and 5. For CRP, S was of 93% with SP of 20%, E of 38%, PPV of 27%, and NPV of 91%. The remaining parameters showed deficient correlation with CLINIC. Correlations between CD64 and CRP, BANDS, and ANC were of 0.435, 0.342, and 0.01, respectively. Conclusions Neutrophil CD64 expression quantitation provides improved diagnostic detection of infection/sepsis compared with the standard diagnostic tests used in current medical practice. CD64 expression showed a better PPV than CRP, and an acceptable NPV. CRP showed deficient SP and E. BANDS, GI, and ANC showed no correlation with CLINIC. CD64 is a new indicator of infection that deserves consideration to be introduced in the daily hematology laboratory analysis. Disclosures No relevant conflicts of interest to declare.

Description: More aggressive therapies used for treatment of oncohematological malignancies or control of immune responses are resulting in an increased frequency of platelet counts below the 50 x 109/L limit. The recommended reference method for platelet counts was tedious and showed low reproducibility until now. In the last 2 years, flow cytometry based techniques combined with specific monoclonal antibodies (MoAbs) have been accepted as reference method. We have evaluated the accuracy for low platelet counts of several hematologic analyzers currently used in our laboratories. The new reference method approved by ISLH, ICSH y NCCLS is based on double labeling of platelets using MoAbs directed to CD41 and CD61 followed by flow cytometry analysis. Absolute platelet counts are calculated using a ratio with red blod cell (RBC) counts provided by the hematological analyzers. In our studies, 50 blood samples with platelet counts ranging from 1.5 to 39.4 x109/L were processed in duplicate through 1 Advia 2120 (Bayer Diagnostics), 2 Advia 120 (Bayer Diagnostics) and 2 Pentra 120 DX (Horiba-ABX Diagnostics). Advia analyzers use laser-based technology while Pentra analyzers use impedance one for cell counting. All samples were also processed through the reference flow cytometric method, being platelets identified by their double labeling for CD41 and CD61. The minimal number of platelets acquired in the platelet region was established at 1000. Absolute platelet counts were calculated using RBC counts provided by the respective analyzers. All blood samples were processed within 6 hours from phlebotomy. Statistical methods applied included: coefficient of variation (%CV), coefficient of correlation (r ), linear regression, Passing-Bablock (P-B) regression and Bland-Altman test. Precision of each analyzer was obtained by processing in 10 times different blood samples with counts from 4 to 39 x 109/L. Global results were evaluated, though special attention was paid to subgroups of results below or above 20 x 109/L. Correlation between reference values and counts provided by the Advia 2120 was 0.945 with a linear regression of 0.987x+2.9. P-B correlation was good and the average difference was 2.7 x 109/L. In the subgroup of samples with counts below 20 x 109/L correlation was 0.874 with 1.00x+2.7. P-B was correct and the average difference was 2.8 x 109/L. Results with Advia 120 were always similar to those calculated with the Advia 2120, though the average difference was slightly lower with a value of 1.7 x 109/L. Precision (CV) was 16% for platelet count levels at 4 x 109/L, 12% for those at 13 x 109/L and 4% for those at 39 x 109/L. Correlation with Pentra 120 Dx was 0.937 with a linear regression of 0.894x+2.7, the P-B was acceptable with an average difference of 1.2 x 109/L. Correlation index was 0.824 with a linear regression of 0.88x+2.8 for platelet counts below 20 x 109/L, average difference was of 1.4 x 109/L and a correct P-B. Precision (CV) ranged from 26% at 4 x 109/L and 8% at 20 x 109/L platelet counts. Our data demonstrate that hematological analyzers evaluated provided very reliable results at low platelet counts. Advia and Pentra analyzers investigated tend to over calculate the number of platelets (2.5 and 1.4 x109/L respectively). Correlation scattering was slightly superior with the Pentra analyzer. Overall reproducibility for low platelet counts was excellent for both laser and impedance technologies tested.

Description: While procoagulant activity of tissue factor (TF) has been widely investigated, its possible proadhesive properties towards platelets have not been studied in detail. We explored the interaction of platelets with human TF (hTF) firmly attached to a surface using anticoagulated blood with low molecular weight heparin (20 U/ml) at different shear rates. For studies at 250 s−1 and 600 s−1, TF adsorbed on a synthetic surface was exposed to circulating blood in flat perfusion devices. Deposition of platelets and fibrin formation were evaluated by morphometric, immunocytochemical and ultrastructural methods. For experiments at 5000 s−1, we used the PFA-100™ with experimental cartridges with collagen or collagen-hTF. Effect of rFVIIa was assessed in all experimental settings. Prothrombin fragment F1+2 levels were also measured. At 250 and 600 s−1 platelet interaction was 19.84±1.33% and 26.12±3.42% of the total surface respectively. Our inmunocytochemical results suggest that von Willebrand factor could mediate these interactions. Fibrin formation was significantly higher at 250 s−1 than at 600 s−1 (p

Description: Serotoninergic mechanisms are reciprocally implicated in depression and cardiovascular disorders. Serotonin (5-HT) may facilitate the development of a subpopulation of platelets with increased procoagulant activity. We have investigated the involvement of serotoninergic mechanisms in adhesive, cohesive and procoagulant function of platelets. Furthermore, we evaluated the antithrombotic properties of selective serotonin reuptake inhibitors (SSRI). For these purposes we used a series of experimental strategies including standard aggregometry, flow cytometry, perfusion techniques and determination of coagulation parameters. Serotonin concentrations ranging 0.5–5 μM were used in these studies. Citalopram, a selective serotonin reuptake inhibitor (SSRI), was used at concentrations equivalent to those reached in clinical practice. Aggregation studies indicate that 5-HT is a weak agonist for platelets in comparison with standard activating agents. Only the highest concentrations of 5-HT tested (5 μM) caused minimal and reversible platelet aggregation. Despite this modest effect on aggregation, 5-HT potentiated the aggregation induced by low concentrations of ADP (0.5 μM) in PRP samples (16.3±5.9 % vs. 28.1±5.5 %). Potentiation of aggegation was more evident when PRP was obtained from blood anticoagulated with LMWH (p