Clinically feasible method for assessing leukocyte rheology in whole blood
Riha ShimizuHirotsugu FukudaYuji KikuchiHirokazu YanakaNobuhiro HataMasashi YamazakiYuki NakataniYuma TamuraSeiko YamakoshiAtsuhiko KawabeYasuto HorieHiroyuki SugimuraYasushi MatsushitaTakaaki NakamotoTakanori Yasu
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This study reports a novel method for assessment of leukocyte rheological activation with a new designed microchannel array chip to mimic the human microvascular network for microchannel array flow analysis (MCFAN). Study subjects were 79 healthy volunteers and 42 patients with type 2 diabetes mellitus (DM) and 36 patients with acute coronary syndrome (ACS). Using the anticoagulants heparin and ethylene-diamine-tetraacetic acid (EDTA)-2Na which inhibits platelets and leukocytes by chelating Ca2+, we were able to quantify leukocyte rheological activation by the subtraction of passage time of blood treated with both heparin and EDTA-2Na from that of blood treated with heparin only. We confirmed that passage times of whole blood with heparin + EDTA-2Na were always shorter than those of whole blood with only heparin in healthy subjects and patients with DM or ACS under suction pressures of − 30 cmH2O. There was a significant correlation between delta whole blood passage time {(heparin tube) − (EDTA-2Na + heparin)} and serum levels of myeloperoxidase and adhesive leukocyte number, respectively, even in blood from patients with DM or ACS, who suffered from inflammation. In conclusion we have developed a clinically feasible method for assessing leukocyte rheological activation in whole blood in ex vivo.Keywords:
Ex vivo
In this paper,the determination of P selectin expression as a market of platelet activation was demonstrated. Blood was taken by venipuncture without tourniquent with the first two mililiters of blood discarded and the 1 ml sample of whole blood was collected in 0 1 ml of 3 8% sodium citrate.Within 3 minutes each of 5 μl sample of CD42a FITC(to label platelets),CD62P PE(to label activation marker P selectin)and CD45 CY(to label white cells)was added to each of 1 ml sample of whole blood.After carefullu mixing and incubating for 20 minutes at ambient temperature,samples were fixed in paraformaldehyde(1%) for 10 minutes and washed twice with PBS solution.All the speciemens were assayed with lasar flow cytometry.It was found that the positive percentages of P selectin expression on platelet in whole blood were lower than those in platelet enriched plasma(PRP). This method of platelet activation determination appears to be more objective and specific, thus should better reflect the physiological status of platelet activation in vivo.
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White cell (WBC)-reduction filters that remove more than 99 percent of the WBCs from platelet concentrates are rapidly being introduced into routine use. Using activation-dependent monoclonal antibodies and flow cytometry, platelet activation was evaluated before and after WBC reduction in 10 platelet concentrates prepared manually from whole blood obtained from five male and five female regular volunteer blood donors. In general no significant increases were found in platelet activation markers after WBC reduction using filters. However, if platelets were activated during preparation, increased numbers of platelets were found expressing the activation marker CD62, and this correlated with the decrease in the platelet count after WBC reduction. These observations may explain increased platelet loss following WBC reduction in some platelet components.
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Platelets are transfused to patients to prevent bleeding. Since both preparation and storage can impact the hemostatic functions of platelets, we studied platelet concentrates (PCs) with different initial composition in regard to platelet fragmentation and its impact on storage-induced changes in activation potential. Ten whole blood derived PCs were assessed over 7 storage days. Using flow cytometry, platelet (CD41+) subpopulations were characterized for activation potential using activation markers (PAC-1, P-selectin, and LAMP-1), phosphatidylserine (Annexin V), and mitochondrial integrity (DiIC1(5)). Aggregation response, coagulation, and soluble activation markers (cytokines and sGPVI) were also measured. Of the CD41+ events, the PCs contained a median of 82% normal-sized platelets, 10% small platelets, and 8% fragments. The small platelets exhibited procoagulant hallmarks (increased P-selectin and Annexin V and reduced DiIC1(5)). Normal-sized platelets responded to activation, whereas activation potential was decreased for small and abolished for fragments. Five PCs contained a high proportion of small platelets and fragments (median of 28% of CD41+ events), which was significantly higher than the other five PCs (median of 9%). A high proportion of small platelets and fragments was associated with procoagulant hallmarks and decreased activation potential, but, although diminished, they still retained some activation potential throughout 7 days storage.
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Activated clotting time
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Whole blood activated clotting time (ACT) can be determined by many different methods that use a variety of clotting cascade activators and end-points. This study compared the results of three whole blood ACT instruments at equivalent concentrations of heparin. Whole blood (9.8 ml) from 10 healthy adult volunteers without coagulation abnormalities was added to 0.2 ml of heparin solution producing heparin concentrations of 0, 0.1, 0.2, 0.4, 0.6, 0.8, and 1.0 U/ml. Coagulation status was determined in duplicate with the Hemochron 400 System (HC), the HemoTec Automated Coagulation Timer (HT), and the TriMed ACTivator (TM). Thrombin times or dilutions (TT) were also determined for each sample. Baseline values did not differ (p greater than 0.05); however, the HT and TM ACT values were significantly longer (p less than 0.05) than the HC ACT values at predicted heparin concentrations greater than 0.2 U/ml. Results from the HT and TM instruments were not significantly different. The HT and TM instruments both provided a greater ACT range over the heparin concentrations tested. Of the tests studied to monitor heparin therapy, mean scaled TTs showed the best correlation with predicted heparin concentrations.
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Background and Aims: Olive oil consumption is associated with lower cardiovascular disease (CVD) risk. We recently demonstrated that frequent olive oil consumption was inversely associated with thrombin-induced platelet activation ex vivo . While various olive oil phenolic compounds, notably hydroxytyrosol, have been shown to reduce human platelet aggregation ex vivo , little is known about how olive oil affects platelet activation, a key mediator of atherothrombosis. Therefore, we sought to investigate whether hydroxytyrosol exposure affects platelet activation. Methods and Results: Ten healthy, non-smoking men and women (25.0±1.6 years) with normal body mass index (BMI 22.2±3.3 kg/m 2 ) were recruited for ex vivo platelet studies. Individuals on antiplatelet or anticoagulation medications or medications known to influence lipid levels were excluded. Subjects attended a single visit at NYU Langone where platelet-rich plasma (PRP) was isolated and incubated with three different concentrations of hydroxytyrosol or ethanol vehicle for 60 minutes at room temperature. PRP was then incubated with bovine thrombin for 5 minutes followed by anti-CD62P for 15 minutes in the dark. Platelet activation was determined by platelet surface expression of P-selectin, assessed via flow cytometry. E x vivo exposure to hydroxytyrosol inhibited thrombin-induced platelet P-selectin expression in a dose-dependent manner (Figure). Conclusions: We demonstrate that hydroxytyrosol exposure reduces thrombin-induced platelet P-selectin expression in a dose-response fashion. The dose response seen in this ex vivo platelet activation study is consistent with our clinical observation that more frequent olive oil intake was associated with reduced ex vivo thrombin-induced P-selectin expression. This laboratory study suggests a putative mechanism underlying our clinical observation as well as the established inverse association of olive oil consumption with CVD.
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Background and Objectives: Hyperconcentration of platelets may lead to platelet activation and loss of platelet function. Materials and Methods: Platelet activation following hyperconcentration was assessed using flow-cytometric detection of platelet P-selectin expression and platelet swirling. Results: Platelet hyperconcentration led to a minimal increase in P-selectin expression and no differnce in platelet swirling. Conclusion: Hyperconcentration was not associated with a clinically significant change in platelet activation and had no significant effect on platelet quality as detected by pH and platelet yield.
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Abstract Background and Objectives: Hyperconcentration of platelets may lead to platelet activation and loss of platelet function. Materials and Methods: Platelet activation following hyperconcentration was assessed using flow‐cytometric detection of platelet P‐selectin expression and platelet swirling. Results: Platelet hyperconcentration led to a minimal increase in P‐selectin expression and no differnce in platelet swirling. Conclusion: Hyperconcentration was not associated with a clinically significant change in platelet activation and had no significant effect on platelet quality as detected by pH and platelet yield.
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Platelet function was studied in CPD whole blood stored at 4°C for one and three days and in platelet concentrates stored at room temperature for the same periods of time. Comparisons were made of platelet shape, nucleotide content, β-thromboglobulin (βTG) liberated during storage, and platelet aggregation in response to ADP, collagen, sodium arachidonate and ristocetin. It was found that in whole blood the shape of the platelets was less discoid than in platelet concentrates. However, platelet aggregation in response to ADP, collagen, and sodium arachidonate was preserved better in whole blood than in platelet concentrates. Platelet nucleotides were the same in whole blood as in platelet concentrates, but the plasma levels of βTG were less in whole blood. The results show that as judged by aggregation, βTG release and nucleotide content, platelets from whole blood were at least as functional as those from platelet concentrates. However, platelets from whole blood had lost their discoid shape, which suggests that they would have a short survival in the circulation.
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