Filtered platelets

The assay, introduced by Bom (1962a,b), has become a standard method in clinical diagnosis of platelet function disorders and of aspirin intake. Furthermore, the method is used in the discovery of antiplatelet drugs with the advantage of rapid measurement of a functional parameter in intact human platelets. However, processing of platelets during the preparion of PRP, washed or filtered platelets from whole blood results in platelet activation and separation of large-size platelets. 

Blood is drawn from healthy adult volunteers, who had no medication for the last two weeks. Venous blood (8.4 ml) is collected into 1.4 ml ACD-solution and centrifuged for 10 min at 120 x g. The platelet-rich plasma (PRP) is carefully removed, the pH adjusted to 6.5 with ACD-solution and centrifuged at 285 x g for 20 min. The resulting pellet is resuspended in Tyrode s buffer (approx. 500 xl buffer/10 ml PRP). The platelet suspension is applied immediately to a Sepharose CL 2B column equilibration and elution at 2 ml/min flow rate is done with Tyrode s buffer without hirudin and apyrase. Platelets are recovered in the void volume. Final platelet suspension is adjusted to 4 x 108/ml. Gel-filtered platelets (GFP) are kept at room temperature for 1 h until the test is started. 

Thrombin-induced gel-filtered platelet aggregation assay. 

The change in biological response of the adsorbed fibrinogen molecule (conversion), is also noticeable with platelet adhesion studies. In confirmation of earlier studies of Zucker and Vroman (5), we found that, usually, less platelets adhered to areas of glass slides exposed to platelet-poor plasma for 3 min than areas exposed for 3 s. When, however, a gel-filtered platelet suspension was used in place of platelet-rich plasma, a dramatic difference in the number of platelets attached to the surface previously exposed to platelet-poor plasma for 3 s or 3 min occurred. Therefore, this more reproducible protocol was used to study not only the adhesion of platelets onto artificial surfaces but also as a probe of conversion. For this purpose we chose a series of block copolymers with controllable domain morphology (phase separation on a molecular scale) and different surface energies (wettability). Previous studies have shown that the degree of phase separation influences the interactions with blood components (6,7). 

Figure 7. (A) Dose response of Fg-Fab 2(E) polymer-induced aggregation of gel-filtered platelets. Values indicated are final concentrations of the polymer. (B) Aggregation of gel-filtered platelets by Fg-Fab 2(E) polymer and lack of aggregation by its components. Aggregation in response to Fg-Fab 2(E) polymer (150 ug/ml) or fibrinogen (500 ug/ml) followed by ADP (5 uM) is contrasted to the lack of response to Fab 2(E) antibody (300 ug/ml) alone or fibrinogen (500 ug/ml) alone. (Reproduced with permission from Ref. 38. Copyright 1986 Grune Stratton.)...

Figure 8. Effects of various inhibitors on Fg-Fab 2(E) polymer-induced aggregation of gel-filtered platelets, (Reproduced with permission from Ref. 38. Copyright 1986 Grune Stratton.)...

Figure 1.1 Average increase in free fatty acids induced by thrombin using the platelets from eight donors. Gel-filtered platelet suspensions were preincubated with 100 fiM BW 755 C for I min at 37°C and then incubated with 5u/ml thrombin for a further 5 min at 37°C. Free fatty acids were extracted and analysed as methyl esters by GLC Reproduced from Smith et al, Biochim. Biophys. Acta, 835, 344, 1985 with permission...

Shiba, M., Tadokoro, K., Sawanobori, M., Nakajima, K., Suzuki, K., Juji, T Activation of the contact system by filtration of platelet concentrates with a negatively cheu-ged white cell-removal filter and measurement of venous blood bradykinin level in patients who received filtered platelets. Transfusion 37,457-462 (1997)... 

Filtration Filtration (qv) is appHed in blood cell separation to remove leukocytes from ted blood cell (RBC) and platelet concentrates. Centtifugational blood cell separators do not reduce white blood cells (WBC) in red cell and platelet products sufficiently to avoid clinical complications such as GvHD and alloimmunization. A post-apheresis filtration step is needed to further reduce the WBC load. Modem filters are capable of a 3-log reduction in white cell contamination of the blood product, eg, apheresis single-donor platelet units having a typical white cell contamination of 5 x 10 white cells in 4 x 10 platelets can be reduced to a 5 x 10 white cell contamination, a sufficiently low number to avoid severe transfusion reactions. 

Surface Tension. Interfacial surface tension between fluid and filter media is considered to play a role in the adhesion of blood cells to synthetic fibers. Interfacial tension is a result of the interaction between the surface tension of the fluid and the filter media. Direct experimental evidence has shown that varying this interfacial tension influences the adhesion of blood cells to biomaterials. The viscosity of the blood product is important in the shear forces of the fluid to the attached cells viscosity of a red cell concentrate is at least 500 times that of a platelet concentrate. This has a considerable effect on the shear and flow rates through the filter. The surface stickiness plays a role in the critical shear force for detachment of adhered blood cells. 

Cell Activation. Several studies have shown that platelets and white cells undergo shape changes when adhering to filter media. The cells are activated by contact with the filter media and form pseudopods which attach to the filter media. The cells membranes may need a certain degree of viabihty to be able to actively attach to the filter media. When white cells are treated with metaboHc inhibitors, the capabiUty of leukocyte reduction by the filter is reduced. 

Cell Adhesion. The membranes of leukocytes and platelets contain a variety of components that promote ceU-surface contact. Although numerous ceU-surface molecules are likely to play a role in ceU-surface adhesion, the group of selectins are of particular interest to research on this subject. Selectins are molecules that are known to promote leukocyte—platelet adhesion. However, selectin-based models have not been able to account for the fact that platelets are allowed to pass through the filter and leukocytes are not. 

Cell—Cell Interactions. Older generations of leukocyte filters depended partly on the formation of platelet—leukocyte—thrombin formations. It is not clear whether this mechanism plays a role in third-generation filters. 

The -toluenesulfonylhydrazide separates as fluffy, white platelets. The hydrazide, after cooling overnight in a refrigerator to complete crystallization, is filtered through a Buchner funnel. The filter cake is washed several times with petroleum ether (b.p. 35-60°), sucked dry, and then air-dried. / -Toluenesulfonyl-hydrazide (172-173 g., 90% yield) is obtained as a white, odorless, crystalline product, m.p. 101-104° (Note 7). 

The eluate is concentrated to dryness under reduced pressure, taken up in 25 cc of hot acetone, filtered, and diluted with ether. The pigment which crystallizes as red-brick colored platelets is essentially pure but may be recrystallized if desired from hot ethyl acetate. An analysis of the product showed C = 59.01 H = 6.B1 N=13.3B. 

The methionine nitrile (20 g) is dissolved in a solution prepared from 50 ml of aqueous 5N sodium hydroxide solution and 65 ml of ethanol. The solution is then refluxed for 24 hours ammonia is evolved. The solution is treated with activated carbon, filtered, acidified with glacial acetic acid (17 ml), chilled to -10°C and filtered to give crude product. This crude product is then slurried with a solution made up of 20 ml of water and 20 ml of methanol, filtered at -5° to -H0°C and dried to give dl-methionine as white platelets. 

C with a solution of 16 g of sodium nitrite in 40 ml of water. To this solution was added a solution of 8.5 g of cyanoacetic acid in 100 ml of water cooled to 0 °C. The resulting solution was neutralized with 200 ml of a 20% solution of sodium hydroxide when an intense red color formed. The formazan was precipitated with 10% hydrochloric acid, filtered, and air dried. The crude formazan was recrystallized from 400 ml of ethanol to yield 9.5 g (40%) of golden platelets mp 158 °C. 

Figure 5.25 — Flow-through ion-selective optrode based on a multilayer lipidic membrane prepared by the Langmuir-Blodgett method. (A) Cross-sectional view of the composite six-layer membrane (four layers of arachidic acid/ valinomycin covered by an arachidic acid and rhodamine dye bilayer). (B) Optical arrangement integrated with the sensor, which is connected to a flow system. LS light source Ml and M2 excitation and emission monochromator, respectively FI and F2 primary filters M mirror LB lipid-sensitive membrane in a glass platelet FC flow-cell A amplifier D display P peristaltic pump. (Reproduced from [107] with permission of the Royal Society of Chemistry).

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