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In vivo platelet activation results are often confounded by activation induced in vitro during the preparative procedures. We measured ex vivo (basal) and in vitro (thrombin-induced) platelet activation in sodium citrate, ethylenediaminetetraacetic acid (EDTA), and Citrate Theophylline Dipyridamole Adenosine (CTAD) whole blood specimens. Determinations were made by measurements of platelet density (mean platelet component: MPC concentration) on the Advia 120 Hematology System. The MPC has been previously shown to correlate with a fluorescence flow cytometric method, also determined in this study, using the surface expression of CD62P. Moreover, platelet shape and structure changes in EDTA and CTAD anticoagulated whole blood specimens were characterized by transmission electron microscopy (TEM). Observations made using the Advia 120 Hematology System platelet density parameter, MPC, in the absence of thrombin were 25.7 +/- 0.9 g/dl, 27.9 +/- 0.9 g/dl and 24.8 +/- 1.2 g/dl in sodium citrate, EDTA and CTAD whole blood specimens, respectively. Addition of thrombin induced a significant change in platelet MPC for sodium citrate (21.9 +/- 1.9 g/dl; p<0.0001) and EDTA (23.2 +/- 0.9 g/dl; p<0.0001) whole blood specimens. In contrast, thrombin had no effect on MPC measured in whole blood taken into CTAD tubes. In vitro fluorescence flow cytometric platelet activation experiments measuring the percentage of platelets expressing anti-CD62P showed increase in sodium citrate specimens from 9.2 +/- 7.0 to 55.5 +/- 23.1 % (p<0.0001) and in EDTA specimens from 1.9 +/- 1.7 to 64.6 +/- 12.4 % (p<0.0001) after addition of thrombin. However, in blood taken into CTAD tubes, there was no significant change. Studies on platelets isolated from whole blood in CTAD showed activation by thrombin indicating that platelets in CTAD, while protected in its presence remained functional upon its removal. When observed by TEM over time, platelets in EDTA appear more activated and contain fewer granules than platelets in CTAD. We conclude that CTAD demonstrates in vitro platelet activation inhibition and may be useful in stabilizing ex vivo platelet activation. The novel platelet activation parameter, MPC, measured by an automated routine hematology system, using customized proprietary software, may be used in conjunction with CTAD, a stabilizing anticoagulant, to measure the ex vivo platelet activation state in whole blood specimens. TEM studies verify shape modifications and simultaneous retention of intracellular granules at early post-venipuncture time periods in CTAD specimens.

The measurement of platelet activation is very difficult to accomplish clinically as platelets are readily activated by in vitro manipulations. Although techniques such as platelet aggregation and flow cytometry exist to estimate platelet function, important limitations prevent these techniques to be widely accepted. In this study, low-fouling surfaces used to limit ex vivo platelet activation were locally bioactivated to rapidly detect platelet activation from whole blood through the selective local adhesion and aggregation of artificially activated platelets. To achieve this result, a fabrication method was developed to create arrays of anti-CD62 and anti-CD61 proteins covalently immobilized on substrates covered by low-fouling graft layers. Moreover, to further limit ex vivo platelet activation and to obtain reproducible results, a custom-made flow chamber was designed and fabricated with the help of computer-assisted mathematical modeling to create defined shear environments. This diagnostic instrument has the potential to allow the rapid estimation of platelet activation levels in whole blood.

Mantle cell lymphoma (MCL) mainly affects elderly and long term response to standard chemotherapy is deceiving. The addition of Rituximab or high dose chemotherapy regimens may improve these results, but have not been evaluated in elderly patients or those with significant comorbidities. We thought to evaluate the safety and efficacy of a High dose Aracytine (Cytosine arabinoside), Rituximab and Dexamethasone (HARD) chemotherapy regimen in elderly patient with aggressive mantle cell lymphoma. Patients and methods: All patients with relapsing, refractory or de novo aggressive MCL were eligible despite comorbidities. All received Rituximab 375 mg/m2 and Dexamethasone 40 mg/d x3 days per cycle. The dose of Aracytine was modulated according to the general condition and accompanying comorbidities. Revised data included clinical and biological parameters, pathology specimens, CT and PET scans. Comorbidities were assessed according to medical records. Patients who received at least one cycle of HARD were analysed for tolerance; and those who completed scheduled treatment or with treatment interruption due to intolerance or insufficient response were analysed for efficacy. Results: Between November 2005 and August 2007, eight consecutive MCL patients, 5 males, 3 females; median age 72.6 years (65–77) were treated by HARD based chemotherapy. All presented with aggressive histological pattern and stage III/IV disease. The majority had performance score >2 (6/8), high serum LDH (6/8), high β2microglobulin (5/5) and Hemoglobin level <12 g/dL (5/8). The FLIPI score was High/High-Intermediate in seven patients. The Charlson comorbidity score was >5 for seven patients and principal documented comorbidities were coronary heart disease (3/8), cardiac systolic dysfunction (3/8), cerebrovascular diseases (3/8), chronic renal insufficiency (4/8), and chronic respiratory disorders (3/8). A total number of 27 HARD cycles were given. All received planned doses of Rituximab and dexamethasone. The median initial dose of Aracytine was 2.55 g/m2 (1.12–4 g/m2), which was subsequently modified according to tolerance. Five patients also received at least one cycle of concomitant Cisplatinum (average dose 20–60 mg/m2). Primary prophylactic G-CSF was given to 5 patients. There were five episodes of thrombopenia and five episodes of anemia requiring transfusions. One patient developed acute transfusion reaction complicated by myocardial infarction, pulmonary edema and acute renal failure that responded to medical treatment. One cycle of HARD was complicated by febrile neutropenia. There was no treatment associated mortality. Two patients are still under treatment and are only evaluated for tolerance. Two other patients were refractory to one and two cycles of HARD and died rapidly from disease progression. Of note that both had >150 x109circulating lymphoma cells/L at treatment. Four patients (67%) responded favourably: 3 complete remissions maintained with 22, 14 and 10 months of follow-up; and one partial remission that persisted for 15 months before progression. Conclusion: HARD is safe in elderly and fragile patients with MCL, provide that the dose is modulated according to comorbidities. Despite this dose modulation, the response rate seems very interesting and compares favourably with other regimens adapted for young and physically fit patients.

INTRODUCTION: Hemostasis is greatly perturbed following coronary arteries surgery with cardiopulmonary bypass (CPB). During off-pump coronary arteries bypass grafting (OPCABG), because of the lack of contact between the blood and the non-endothelial surfaces of the CPB circuit, one might expect that hemostatic defect could be reduced. Recent data (1,2,3,4) even suggest a hypercoagulable state following OPCABG. We addressed this issue by studying coagulation factors, fibrinolysis and hypercoagulability in ten patients who had OPCABG. METHODS: The study protocol was approved by the ethical committee of our institution and consent was obtained from patients the night before their surgery. In 10 patients who had OPCABG, D-Dimers, Factor VII, fibrinogen and prothrombin fragment1.2 (PF 1.2) were assessed at induction of anesthesia, at the end of surgery, 24 hours and 96 hours after surgery. Patients received heparin to maintain an ACT around 250s during surgery and had protamine reversal at the end of surgery. Specimens anticoagulated with sodium citrate 3,2% were centrifuged in less than 1 hour at 4800 rpm for 15 minutes. The plasma was collected and immediately cooled to -80C until analysis. We used analysis of variance and Fisher's PLSD test for statistical analysis. RESULTS: Fibrinogen decreased at the end of surgery and increased at 96 hours, Factor VII decreased in the first 24 hours but returned to normal at 96 hours, PF1.2 increased significantly at 96 hours after the surgery and there was a trend for D-Dimers to increase 24 hours and 96 hours after the surgery (Table). Blood loss during the surgery was 2623 ±1671 ml. DISCUSSION AND CONCLUSIONS: Prothrombin fragment 1.2 is a marker of systemic thrombin generation and its increase represents a procoagulant state. In the first day following OPCABG surgery, there is decrease of coagulation factors (fibrinogen and factor VII). Hemodilution probably account for these results as our patients had important volemic shifts during their surgery. Consumption of factor might be a minor contributor but systemic activation of coagulation was not present at this time. However, at 96 hours after surgery, PF 1.2 was significantly increased, factor VII returned to normal and fibrinogen increased. A systemic inflammatory response can be observed after most major surgeries. The increase in fibrinogen at 96 hours in our patients is probably related to this inflammatory response as might be the hypercoagulable state observed. The factor VII is not know to be a marker of inflammation and returned to normal. Thus our results support a hypercoagulable state after OPCABG.