Platelet-poor plasma

In vitro platelet activation is dependent on the anticoagulant that is used for blood collection. In one study it was demonstrated that PF4 levels in platelet-poor plasma isolated after incubation without any stimuli for 1 hour at 37°C were as follows conventional heparin, 1180 ng/ml hirudin, 469 ng/ml citrate, 440 ng/ml and EDTA, 217 ng/ml (110). EDTA appears to suppress platelet degranulation. PF4 levels obtained with a low-molecular-weight heparin preparation called Frag-min were, however, comparable to those obtained with hirudin (110). 

The accumulation of di(2-ethylhexyl) phthalate in platelet-poor plasma stored for seven and 14 days in PVC bags sterilized by steam, ethylene oxide or irradiation revealed seven-day storage levels of di(2-ethylhexyl) phthalate of 378 19, 362 10 and 275 15 mg/L, respectively, and 14-day storage levels of 432 24, 428 22 and 356 23 mg/L, respectively (Dine et al., 1991). 

Contreras TJ, Sheibley RH, Valeri CR. 1974. Accumulation of di-2-ethylhexyl phthalate (DEHP) in whole blood, platelet concentrates and platelet-poor plasma. Transfusion 14 34-46. 

The entire procedure is performed in plastic (polystyrene) tubes and carried out at room temperature. Freshly collected venous blood is anticoagulated with hirudin (1 volume + 9 volumes of animal blood) or ACD solution (1 volume + 9 volumes of human blood) and centrifuged at 170 x g for 15 min to obtain platelet-rich plasma (PRP). The PRP-supematant is carefully removed, and the rest is further centrifuged at 1500 x g for 10 min to obtain platelet-poor plasma (PPP). PRP is diluted with PPP to a platelet count of 3 x 108/ml before use in the aggregation assays. To obtain washed platelets (WP), 8.5 volumes of human blood are collected into 1.5 volumes of ACD and centrifuged as for PRP. PRP is acidified to a pH of 6.5... 

Blood from healthy human volunteers was collected into plastic vessels containing 3.8% sodium citrate (1/10 volume) and platelet-rich plasma obtained from the supernatant fraction of blood after centrifugation. Platelet-poor plasma was obtained by centrifugation of the remaining blood. 

Platelet-poor plasma is prepared from venous blood by centrifugation at 1000 xg for 15 min, and an equal volume of ethanol is added. The mixture is left on ice for 10 min and then centrifuged at 6500 xg for 15 min. Cyclooxygenase activity is measured polarographically, using an oxygen electrode and an incubation buffer (total volume 3 ml) containing potassium phosphate (pH 7.2, 0.1 M), 100 pM arachidonic acid, 1 mM phenol and 2.5 mM sodium cyanide. The reaction is... 

Platelet rich and platelet poor plasma (PRP and PPP) apparatus to measure platelet aggregation (dual channel ag-gregometer, model DP-247E, Sienco, Morrison, CO Tur-bidometer, NKK HEMA TRACER I, Niko Bioscientific Co., Tokyo, Japan). 

In a subsequent study, Zucker and Woodard (Z2) prepared what they termed platelet-poor plasma by collecting the blood sample in silicone-... 

The remaining half of the sample collected in the silicone-treated glassware was transferred to ordinary glass and allowed to clot at room temperature for 30 minutes, as is usually done to obtain serum for acid phosphatase determinations. The mean values and their standard deviations for serums from platelet-poor plasma from various groups of subjects were as follows in Bodansky units 28 normal women, 0.094 0.009 23 normal men, 0.109 0.021. The corresponding values for ordinary serum acid phosphatase were 0.226 0.13 and 0.278 0.27. It appeared, therefore, that approximately 60% of the acid phosphatase in serum arises from the liberation of this enzyme from platelets as a result of clotting. 

Serotonin can be measured in whole blood, serum, platelet-rich plasma, platelet-poor plasma (i.e., platelet-free plasma), isolated platelet pellets, urine, and CSF. Most blood serotonin is stored in the platelets and is easily released during sample preparation. For whole blood serotonin, venous blood (10 mL) is drawn into a tube containing potassium EDTA as an anticoagulant, gently mixed, placed on ice, and transferred to a storage tube. An aliquot of blood is then removed for a platelet count alternatively, a simultaneous EDTA blood sample can be collected. Antioxidants such as ascorbic acid or metabisulfite are conveniently added to the storage tube when a fluorometric detection system is used. Blood serotonin samples are stored frozen at -20 °C, preferably within 2 hours after collection. 

Platelet-rich plasma samples are prepared from whole blood by centrifuging at 120g for 30 minutes at 4 or at 200 for 15 minutes at room temperature. To prevent lowering the serotonin concentration, platelet-rich plasma is prepared within 1 hour after the blood is collected and placed on ice. An aliquot of platelet-rich plasma is removed for a platelet count. Platelet-poor plasma and platelet pellets are prepared from measured aliquots of the platelet-rich sample plasma by centrifuging at 4500 for 10 minutes at 4 (or at lOOOg for 30 minutes at room temperature). To reduce the probability of platelet rupture, samples should never be frozen before the cell-free plasma is obtained. Plasma and pellets are stored frozen at -20 °C and analyzed within 1 to 2 weeks after collection. 

Anderson GM, Feibel PC, Cohen DJ. Determination of serotonin in whole blood, platelet-rich plasma, platelet-poor plasma and plasma ultrafiltrate. Life Sd 1987 40 1063-70. 

Ishida J, Takada M, Hitoshi N, lizuka R, Yamaguchi M. 4-Dimethylaminobenzylamine as a sensitive chemiluminescence derivatization reagent for 5-hydroxyindoles and its application to their quantification in human platelet-poor plasma. J Chromatogr B Biomed Sci Appl 2000 738 199-206. 

Middelkoop CM, Dekker GA> Kraayenbrink AA, Popp-Snijders C. Platelet-poor plasma serotonin in normal and preeclamptic pregnancy. Clin Chem 1993 39 1675-8. 

Sodium heparin (Wyeth Lab, Philadelphia, Pennsylvania) was added from a stock solution individually to the fresh platelet-poor plasma samples Just prior to testing. This heparin is derived from porcine intestinal mucosa. 

A 50-mL aliquot of blood collected into 10 mL of anticoagulant (3.8% sodium citrate +2.45% dextrose) was centrifuged at 250 x g for 15 min at room temperature. The platelet-rich plasma was removed and recentrifuged at 1000 x g for 15 min at room temperature. After removing the platelet-poor plasma, the platelet button was suspended in 5 mL of anticoagulant isotonic saline (1 6) to produce a final platelet concentration of at least 500,000/mm3. After exposing the suspension to inIn-oxine (10-15 xg oxine/mL) for 30 min at room temperature, the suspension was centrifuged at 1000 x g for 15 min. The platelet button was washed twice with 1 mL of platelet-poor plasma and finally resuspended in 5 mL of platelet-poor plasma for injection into the animal. 

Figure 2. Composite display of average platelet retention index p for selected polymer types. The acrylates, but not polystyrenes, are significantly passivated by pretreatment with plasma (n = 6). Key , lowest value observed in each case and PPP, preincubated in platelet-poor plasma.

We tested the hypothesis that the platelet retention index should increase as polymer composition varied from 100 mol % methyl acrylate to 100 mol % styrene, the respective platelet retention indices p being about 0.25 and 0.55 for the homopolymers. The results are shown in Figure 3, wherein p increases to a maximum near 40% styrene. When the surfaces were incubated in platelet-poor plasma before contact with whole blood, the values of p were much reduced (from 0.25 to 0.05 for methyl acrylate) for copolymers containing up to 60 mol % styrene. Copolymers of higher styrene content were not rendered significantly less retentive by plasma pretreatment. 

Figure 3. Average platelet retention index p as a function of mole fraction of styrene in a series of copolymers of methyl acrylate and styrene. Key —, control and —, pretreated in platelet-poor plasma.

Preincubation of surfaces with platelet-poor plasma substantially reduced the platelet retention index of polyacrylates and polymethacrylates, but hardly altered the retention index of polystyrene or copolymers with methyl acrylate that are rich in styrene. Even without plasma pretreatment, the surface, when exposed to whole blood, was probably first contacted by molecular elements (including the proteins) before the cellular elements arrived. What then is the mode of action of plasma pretreatment Several hypotheses, none conclusive, can be advanced, such as ... 

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). 

Human platelet-rich and platelet-poor plasmas were obtained from blood drawn by venipuncture into a 10-mL, siliconized Vacutainer (Becton Dickinson) anticoagulated with 0.11M sodium citrate in a 9 1 ratio. All donors had fasted overnight and had not taken medication for at least 2 weeks. Platelet-rich plasma was prepared by placing the anticoagulated blood in polypropylene tubes and centrifuging at 77 g for 10 min at room temperature. Platelet-poor plasma was prepared by centrifuging the blood at 23,000 g for 10 min. Platelet counts were performed using a Coulter counter the platelet-rich plasma had a concentration of 270,000-320,000 platelets/mm3. 

The platelets, at four random fields, were counted visually on a Zeiss Ultraphot II microscope using reflected light and Nomarski optics at a magnification of 1280 X and a field of view of 7542 pan2. At least four samples of each polymer were tested for each experiment, and each experiment was repeated at least once with a different donor. For the glasses, at least 1-2 samples were tested per experiment. Conversion was expressed as the percentage difference between platelet adhesion at 3 s and 3 min of platelet-poor plasma exposure with respect to the 3-s platelet-poor plasma exposure. 

Statistical Analysis. All data for a particular donor were normalized with respect to the hydrophobic glass at 3 min of platelet-poor plasma exposure for that donor, that is, platelet adhesion at 3 min of platelet-poor plasma exposure to hydro-phobic glass was 100%. A one-way analysis of variance was performed, using each material and time of platelet-poor plasma exposure as a variable. For each material and time, the normalized platelet counts for all the donors were summed, and Scheffe s multiple comparisons were performed. For the difference between the two SBS morphologies, a student s t test was used. Data are presented as the mean of the normalized average from each donor and the pooled standard deviation. 

Figure 3. Passivation of polymers of the acrylate series (A) and the methacrylate series (B) by preliminary exposure to platelet-poor plasma (PPP) before whole blood platelet retention vs alkyl side chain length. (Replotted from data in Fig. 5 of Ref. 17.)...

Oxidized fat may also be responsible for the activation of fee coagulation pathway. Autoxidation products of unsaturated fatty acids are potent inducers of thrombin generation in platelet-poor plasma in vitro (64). A nonspecific effect of hydroperoxides could be excluded because rerr-butyl hydroperoxide or cumene hydroperoxide were without effect. An observation highly relevant to this discussion is fee fact that this effect is not seen in chylomicron-free plasma, and the addition of chylomicrons to plasma of fasting subjects increases the thrombin generation markedly (64). 

Platelet Adhesion. Venous blood from healthy human volunteers, who did not receive any agents which affect platelet function, was collected with a vacuum syringe containing 5 % citric acid. The blood was centrifuged at 800 rpm for 10 min at 25 C, and the platelet-rich plasma (PRP) was withdrawn with a polyethylene pipette and placed in clean vials at room temperature. A portion of PRP was diluted by adding PBS and centrifuged at 1800 rpm for 10 min to prepare platelet pellets. The residue of the blood was further centrifuged at 3,000 rpm for 10 min to obtain platelet-poor plasma (PPP). The platelet count was determined with Coulter Counter (Type 4) and adjusted to have 150,000 platelets in 1... 

In this study, we mainly investigated the part of platelet-poor plasma in platelet reactivity and prostaglandin biosynthesis from exogenous and endogenous arachidonic acid. 

The half-life of thromboxane A2 in platelet-poor plasma was determined as described (9). Briefly, radiolabelled thromboxane A2 was generated from human platelets as enzymatic source and immediately dived in the studied plasma. Then, aliquots of plasma were decanted in 80 volumes of methanol to obtain mono-o-methyl thromboxane B2 from thromboxane A2 (10). Derivatized thromboxane B2 was radiochemically assayed (8) and a semi-logarithmic regression leads to half-life determination. 

Thus, isolated platelets were hyperactive to collagen but not to sodium arachidonate. Moreover, platelet-poor plasma induced a higher response of platelets to arachidonate. 

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