Effects of the addition of second-messenger effectors to platelet concentrates separated from whole-blood donations and stored at 4 degrees C or -80 degrees C

BACKGROUND: Platelet concentrates (PCs) are currently stored at 22 degrees C under continuous agitation. Because of the potential risk of the overgrowth of bacteria in case of contamination, PC shelf life is limited to 5 days. A mixture of second-messenger effectors is being evaluated to determine if it has benefits for cold liquid storage and cryopreservation of platelets. STUDY DESIGN AND METHODS: PCs separated from whole-blood donations by the buffy coat method were randomly assigned (n = 6 each) to be stored for 5 days at 22 degrees C under continuous agitation or at 4 degrees C after treatment with a platelet storage medium (ThromboSol, LifeCell Corp. ). PCs were also cryopreserved with 6-percent DMSO (final concentration) or with ThromboSol plus 2-percent DMSO (final concentration) (TC). After storage, platelets were analyzed by flow cytometry, transmission electron microscopy, and aggregation and perfusion techniques. RESULTS: Cold liquid storage of ThromboSol-treated platelets resulted in a lower binding of coagulation factor Va on the platelet surface than on platelets stored at 22 degrees C. In transmission electron microscopy, a conversion to spherical morphology was seen in the case of cold liquid storage. No difference between ThromboSol-treated platelets stored at 4 degrees C and platelets stored at 22 degrees C was seen in perfusion studies. Cryopreservation in the presence of TC prevented the reduction in glycoprotein Ib and IV expression on platelet surface that is seen in 6-percent DMSO-cryopreserved platelets. Platelets cryopreserved in TC covered, by thrombus, a significantly greater percentage of the perfused surface after the freezing and thawing process. CONCLUSION: ThromboSol-treated PCs separated from whole-blood donations by the buffy coat method, stored at 4 degrees C for 5 days, or cryopreserved in the presence of TC, maintained in vitro functional activity comparable to that achieved by current methods of storage, although discoid morphology was not preserved during cold liquid storage with ThromboSol.     

Recovery of in vitro functional activity of platelet concentrates stored at 4 degrees C and treated with second-messenger effectors

BACKGROUND: The potential for bacterial contamination limits the storage of platelets at 22 degrees C to 5 days. Refrigerated storage at 4 degrees C would abrogate this problem but would also result in a rapid loss of in vitro viability and functional activity and in vivo viability. The inhibition of platelets during storage by a combination of specific, reversible, second-messenger effectors has been investigated to allow prolonged storage at 4 degrees C with significant retention of in vitro viability and functional activity. STUDY DESIGN AND METHODS: The combination of effectors was added directly to platelet concentrates, and this step was followed by storage at 4 degrees C. Control units were incubated at 4 degrees C without the effectors and at 22 degrees C according to standard blood-banking techniques. At 1, 5, and 9 days, the units were tested for recovery of cell number, recovery of in vitro functional activity and viability, and expression of platelet surface markers. RESULTS: Treated platelets stored at 4 degrees C for 9 days, while spherical in shape, displayed no loss of cell number and had a recovery of viability and functional activity, as compared with control platelets stored at 22 degrees C for 5 days, as follows: ADP and collagen aggregation responses of 250 and 100 percent, respectively; a 70-percent recovery of hypotonic shock response; and a 60-percent recovery of extent of shape change. The treated platelets also expressed an equivalent amount of the surface marker glycoprotein lb and a lower amount of the activation marker alpha-granule membrane protein-140 on the membrane surface. CONCLUSION: Second-messenger effectors added to platelets significantly maintained in vitro functional activity with storage at 4 degrees C. In vitro analysis demonstrates the potential for extended 4 degrees C storage of platelets with numerical and functional recovery comparable to that achieved with current methods. Refrigerated storage of platelet concentrates has the potential to reduce the risk of bacterial contamination.     

Effect of platelet activation inhibitors on the loss of glycoprotein Ib during storage of platelet concentrates

Platelet membrane glycoprotein Ib (GPIb) in stored platelet concentrates was analyzed by flow cytometry with three separate monoclonal antibodies (AN-51, 6D1, and SZ-2), by tritiated glycoprotein radiolabeling, and by ristocetin-induced agglutination. Flow cytometry showed that a population of surface GPIb-negative platelets was evident at 5 days and increased three- to fivefold by the tenth day. Tritium radiolabeling of surface GPIb showed a decrease over 10 days of 37% +/- 17%. The degree of loss of surface GPIb correlated well with other changes during storage: decreased ristocetin-induced agglutination, decreased responsiveness in the hypotonic shock test, lower plasma pH, and increased extracellular lactic dehydrogenase. Immunoblotting of total platelet GPIb with the SZ-2 antibody showed a decrease of 58% +/- 16% during the 10-day storage period. The effect of protease inhibitors or platelet activation inhibitors on the loss of GPIb during storage was studied in platelet concentrates paired with untreated controls. Only the platelet activation inhibitors prostaglandin E1 and theophylline retarded the loss of surface GPIb levels (93% +/- 5% GPIb remaining vs 65% +/- 16%). Total GPIb levels also decreased less in the presence of the activation inhibitors (45% +/- 22% lost vs 70% +/- 14% lost). These findings suggest that platelet activation, rather than plasma enzymatic activity, is responsible for the loss of platelet GPIb during storage of platelet concentrates.     

Quality assessment of platelet concentrates supplemented with second-messenger effectors

BACKGROUND: While reducing the potential for bacterial contamination, the storage of platelet concentrates (PCs) at refrigerated temperatures is not routine, because of the induction of the so-called platelet storage lesion. As the modulation of second-messenger levels might help to overcome this drawback, a quality assessment of PCs treated with a mixture of second-messengers effectors known as ThromboSol was performed. STUDY DESIGN AND METHODS: The PCs were supplemented with ThromboSol or phosphate-buffered saline, and stored in parallel at 22 degrees C with continuous agitation or at 4 degrees C. At 1, 5, and 9 days, an in vitro quality assessment of the PCs was performed, including measurement of cell number, metabolic and integrity markers, platelet surface expression of glycoproteins, platelet response to ristocetin and thrombin, and levels of cyclic adenosine 3', 5' monophosphate (cAMP) and thromboxane B2 (TxB2). RESULTS: Control PCs stored at 4 degrees C underwent aggregation and displayed a significant decrease in the platelet number (40% on Day 5). By contrast, the ThromboSol-treated PCs maintained 80 percent of their initial platelet concentration after 9 days of storage at 4 degrees C. Compared to PCs stored at 22 degrees C, refrigerated PCs exhibited minor changes in metabolic values throughout storage, but the addition of ThromboSol induced a rise in metabolic rate during storage at 22 degrees C. Platelet responsiveness to both ristocetin and thrombin was maximally preserved in the ThromboSol-treated PCs stored at 4 degrees C. These units also maintained high levels of cAMP and low concentrations of TxB2 during storage. CONCLUSION: The pharmacologic supplementation of PCs with ThromboSol significantly favors the maintenance of in vitro integrity and responsiveness of platelets during extended storage at refrigerated temperature. This protective effect seems to be a consequence of the ability of ThromboSol's components to sustain high levels of cAMP and to inhibit TxB2 production during the entire extended-storage period.     

Accumulation of DI-2-Ethylhexyl Phthalate (DEHP) in Whole Blood, Platelet Concentrates, and Platelet-Poor Plasma

The concentration of the plasticizer, di-2-ethylhexyl phthalate (DEHP), in the plasma was measured after storage of the whole blood in polyvinylchloride plastic bags at 4 C for up to 38 days in either ACD or CPD. The plasma from ACD-stored whole blood contained more DEHP than that from CPD-stored whole blood. The continuous-flow centrifugation washing procedures removed about 98 per cent of the DEHP from ACD whole blood stored for 33 days at 4 C.
DEHP was assayed in the platelet-rich plasma, platelet-poor plasma, supernatant of the platelet concentrate, washed platelets, and washed red blood cells prepared from CPD whole blood. Very little DEHP was found in the washed red blood cells and platelets, a small amount was found in the platelet-poor plasma, and a large amount in the supernatant of the platelet concentrate. A greater amount of DEHP accumulated in the platelet concentrates that were stored at 22 C than in those stored at 4 C. When platelet concentrates from CPD whole blood were stored at 22 C for 72 hours, the amount of DEHP was about four times that observed after 4 C storage for the same length of time.     

Platelet storage results in a redistribution of glycoprotein Ib molecules. Evidence for a large intraplatelet pool of glycoprotein Ib.

Platelet membrane glycoprotein (GP) Ib contains receptor for von Willebrand factor and thrombin. Its proteolytic fragment, glycocalicin, circulates in normal plasma. In this study, storage of platelet concentrates for 5 d resulted in a 221% increase in plasma glycocalicin (1.3 times the total amount of glycocalicin present on the surface of all platelets), an 8% overall increase in platelet surface GPIb, and the appearance of a surface GPIb-negative subpopulation of platelets. Total platelet GPIb content of fresh washed platelets, determined by gel electrophoresis and immunoassay of Triton X-100 lysates, averaged 159,740 molecules per platelet. There were 36,360 surface GPIb molecules per platelet, determined by immunoassay of the supernatant of fresh washed platelets whose surface GPIb had been completely plasmin-cleaved. In summary, these studies provide evidence for (a) a redistribution of GPIb molecules with platelet storage, and (b) a large intraplatelet pool of GPIb (approximately threefold larger than the platelet surface pool of GPIb).     

Effects of prestorage white cell-reduction of apheresis platelets on platelet glycoprotein Ib and von Willebrand factor

Twenty plateletpheresis components were harvested from 11 healthy donors and stored in polyolefin bags on a horizontal flatbed agitator at 22 degrees C. After 24 hours, white cells were reduced in one aliquot by centrifugation while the other aliquot was stored unaltered. Samples were obtained aseptically from each of these platelets at intervals for up to 10 days, and measurements were made of platelet glycoprotein Ib (GPIb) by both flow cytometry and polyacrylamide gel electrophoresis, of ristocetin-induced platelet aggregation by impedance aggregometry, and of plasma and platelet von Willebrand factor (vWF) by enzyme-linked immunosorbent assay. Storage of platelets under these conditions was associated with only minor decreases in surface GPIb, intraplatelet vWF, and ristocetin-induced platelet aggregation, and no differences were observed between the white cell-reduced and nonreduced aliquots. No benefit of white cell reduction in such components before prolonged storage is evident in the vWF-platelet interaction.     

Cryopreservation of single-donor platelets with a reduced dimethyl sulfoxide concentration by the addition of second-messenger effectors: enhanced retention of in vitro functional activity

BACKGROUND: The potential for bacterial contamination limits the storage of platelets at 22 degrees C to 5 days. This creates an inventory problem, which could be overcome by the use of cryopreservation to allow long-term storage of platelets. It has been demonstrated that the addition to platelets of a mixture of second- messenger effectors (platelet storage solution), allows these cells to retain significant in vitro functional activity following cold storage. Analysis is needed of the ability of this second messenger effector mixture both to protect platelets during cryopreservation and to reduce the need for a cryoprotectant. STUDY DESIGN AND METHODS: Fresh single- donor platelet units (n = 8) were divided into three samples and treated with 6-percent dimethyl sulfoxide (DMSO), 2-percent DMSO or the platelet storage solution and 2-percent DMSO. The samples were placed directly into a -80 degrees C freezer and stored for 1 week, after which they were thawed and analyzed for in vitro functional activity. RESULTS: Platelets cryopreserved with the platelet storage solution and 2-percent DMSO displayed statistically higher retention of functional activity and viability–including cell number, percent of discoid cells, extent of shape change, and hypotonic shock response–than did platelets stored by the method using 6-percent DMSO. In addition, the treated platelets displayed statistically lower expression of p- selectin. The treated platelets showed no loss of cell number, > 88- percent retention of discoid morphology, and > 75-percent retention of ristocetin-induced aggregation as compared to values for these measures in fresh platelets. CONCLUSION: The use of this platelet storage solution in the cryopreservation of platelets yields a significant improvement in their postthaw in vitro recovery and allows for a reduction of the DMSO concentration from 6 to 2 percent, with superior maintenance of in vitro viability and function.     

The effect of platelets and red cells on granulocytes stored at 22 degrees C

Granulocyte concentrates contain varying numbers of platelets and red cells depending upon the method of collection. Either platelet or red cell concentrations may be as high as 2.0 × 10(12) per I. Studies were done on unwashed and washed granulocyte concentrates and on pure granulocyte suspensions with known numbers of platelets or red cells added. These suspensions or concentrates were stored for 72 hours at 22 degrees C. In both experiments, the following were measured: leukocyte and absolute granulocyte counts, dye exclusion, chemotaxis, plasma glucose, plasma pH, and osmolality. Red cell contamination did not adversely effect granulocyte storage. Platelets, however, did contribute to the functional deterioration of stored granulocytes. In the presence of high concentrations of platelets, both granulocyte dye exclusion and chemotaxis were adversely affected at 48 hours of storage. In another experiment, fresh autologous granulocytes incubated for 18 hours in hydroxyethyl starch-citrated-plasma obtained from stored granulocyte concentrates showed a progressive decrease in chemotaxis related to the age of the stored plasma. Glucose supplementation of the spent plasma maintained chemotactic activity. Contamination of granulocyte concentrates with other cells and the availability of glucose to granulocytes are variables affecting the short-term liquid storage of granulocytes at 22 degrees C.     

The effect of storage on platelet morphology

Platelet concentrates were stored for one, two or three days at 4 degrees C (unagitated) or at room temperature (unagitated and linearly agitated). After washing the concentrates twice at room temperature and then incubating them for 60 minutes at 37 degrees C, the platelet morphology was investigated by scanning and transmission electron microscopy. Platelets in freshly prepared concentrates were slightly activated, indicated by some pseudopod formation. Platelets stored at 4 degrees C rapidly lost the normal discoid shape. After three days of storage their surface membranes showed extensive folding, they were slightly vacuolated, and had lost most of their granules. Incubation of cold-stored platelets at 37 degrees C did not induce return to the discoid shape. Room temperature storage resulted in reversal of the slight initial platelet activation. After three days of unagitated room temperature storage the platelets were slightly more vacuolated than those stored with agitation. Room temperature storage usually resulted in well-preserved discoid platelets; however, some agitated platelet concentrates stored at room temperature contained a high proportion of odd shaped cells. This finding could not be correlated with pH change. The failure of platelets stored at 4 degrees C to return to the discoid shape after incubation at 37 degrees C could explain their short survival following transfusion. These results also provide a morphologic correlation with the reported slightly better recovery and survival of platelets stored at room temperature with agitation compared with those stored without agitation.     

Effect of amphotericin B and fluconazole on platelet membrane glycoproteins

BACKGROUND : Fever, chills, and reduced platelet recovery may result when platelets are transfused simultaneously with amphotericin B. Amphotericin B reportedly increases the pitting of membranes in stored platelets. STUDY DESIGN AND METHODS : The effects of amphotericin B and another antifungal agent, fluconazole, on platelet membrane glycoproteins (GP) were examined by the incubation of split aliquots of fresh and stored platelet concentrates (PCs) with these drugs for 3 days in storage bags. To determine the effect of storage, PCs were stored for 5 days, and aliquots removed on Days 1 through 5 were placed in platelet storage bags with 4 micrograms per mL of amphotericin B for 2 to 6 hours. Membrane glycoprotein expression was assessed by flow cytometry with fluorescein isothiocyanate-labeled monoclonal antibodies (MoAbs) directed against the following antigens: GPIb (CD42b), CD63 (an activation protein), P-selectin (CD62), and GPIIb/IIIa (CD41a). RESULTS : Amphotericin B produced a concentration-dependent decrease in the surface binding of CD42b MoAb with no consistent changes in the binding of CD41a, CD63, or CD62 MoAbs after a 3-day exposure. Stored but not fresh PCs showed decreased binding of MoAb CD42b after a 6-hour exposure to amphotericin B (4 micrograms/mL). Fluconazole produced no changes. When the binding of MoAb CD42b to permeabilized platelets was used to measure total platelet content, amphotericin B (4 micrograms/mL) decreased MoAb CD42b binding to a similar degree in fresh and stored platelets. Inhibition of aggregation to ADP and collagen and ADP and epinephrine was seen in stored but not fresh PCs. CONCLUSION : Therapeutic levels of amphotericin B resulted in partial loss of total platelet GPIb in fresh and stored PCs, but decreased surface expression of platelet membrane GPIb only in stored platelets. This difference between fresh and stored platelets may be related to the limited reservoir of GPIb available for redistribution to the membrane in the previously stored PCs and may account for the decreased recovery of transfused platelets observed in some patients receiving amphotericin B.     

Autoapheresis and intraoperative blood salvage in oncologic surgery

Platelet activation occurs during the collection, processing and storage of platelet concentrates. The effect of the platelet activation on the functional state of stored platelets remains however undefined. We employed flow cytometric analysis to evaluate the extent of platelet activation and the physiological response to thrombin stimulation of platelets stored for up to five days under routine blood bank conditions. Platelet surface expression of the activation markers CD62 and CD63 was examined, along with modulation of platelet membrane glycoproteins (GP) Ib and IIbIIIa. Platelet dense granule content was determined using a mepacrine uptake assay and the extent of platelet microparticle generation was quantified. Thirteen random-donor platelet concentrates prepared under routine conditions by a platelet-rich-plasma protocol were examined. Platelets were found to be activated following preparation on day 1. Although a gradual increase was seen with increasing storage time, this was not statistically significant for CD62 or CD63 expression, GPIIbIIIa or GPIb modulation or dense granule release; the generation of platelet microparticles did, however, increase with increasing storage time. The characteristic increase in surface expression of CD62, CD63 and GPIIbIIIa and decrease in GPIb and dense granule content in response to thrombin stimulation was observed with all concentrates, but these measures of platelet functional reserve showed decreasing platelet function with increasing storage time. The results indicate that platelets are activated by day 1, likely as a consequence of manipulation during collection and processing, but are not further progressively activated with increasing storage time; they do, however, become relatively hypofunctional with increasing storage.     

Inhibition of cytokine accumulation and bacterial growth during storage of platelet concentrates at 4 degrees C with retention of in vitro functional activity

BACKGROUND : The potential for bacterial contamination limits the storage of platelet concentrates (PCs) at 22° C to 5 days. In addition, storage of platelets under conventional protocols for longer times (> 3 days), in the absence of white cell filtration, has been correlated with incidents of cytokine-associated febrile reaction in recipients. It has been demonstrated that the addition of a reagent mixture of second-messenger effectors allows platelets stored at 4°C to maintain significant in vitro functional activity. Thus, the effects of 4°C storage on the growth of bacteria and the accumulation of cytokines by the white cell fraction of PCs were analyzed to demonstrate the benefits of this refrigerated storage system. STUDY DESIGN AND METHODS : The platelet storage solution was added directly to PCs obtained from the blood bank, and these treated PCs were stored at 4° C without agitation. In parallel, control PCs were stored according to standard blood-banking procedures. On Days 1, 3, 5, and 9, the PCs were measured for the plasma concentrations of cytokines. Treated and control PCs stored at 4°C and 22°C were inoculated with low-titer Staphylococcus aureus, and bacterial growth was measured over a 5-day period. RESULTS : Control PCs displayed a time-dependent increase in the plasma concentration of interleukin 6, interleukin 1β, and tumor necrosis factor α. These conventionally stored PCs also displayed a time-dependent increase in the bacteria titer. In contrast, the treated PCs stored at 4°C displayed no accumulation of the above cytokines in the plasma fraction and no increase in bacteria titer above the initial inoculation. CONCLUSION : The storage of PCs at refrigerated temperatures inhibits the accumulation of white cell-produced cytokines in the PCs, an effect that could alleviate cytokine-associated febrile transfusion reactions The 4°C storage was also bacteriostatic, which indicates that the storage of PCs at that temperature increases safety by decreasing the potential for sepsis. Thus, the ability to store PCs at 4°C may allow extension of the storage limit beyond 5 days.     

改良血小板添加液低温(10℃)保存血小板的动物实验研究

目的对添加海藻糖的血小板添加液替代70%血浆冷藏的血小板进行动物体内输注效果评价。方法采集兔心脏血,按常规方法制备浓缩血小板,将浓缩血小板分3组保存。血浆常温组:添加100%血浆,22℃保存;添加液低温组:添加70%PAS-ⅢM/30%血浆,10℃保存;冰冻保存组:添加100%血浆,-85℃保存。以新鲜血小板为对照,常温血浆保存组在d 3,添加液低温保存组在d 3、7、9,冰冻保存组在d 20复苏后分别检测血小板缺乏兔模型的血小板24 h回收率和生存率。结果除保存9 d的添加液低温组血小板存活率明显低于新鲜血小板存活率(P<0.05)外,血浆常温组、添加液低温组以及新鲜血小板的24 h回收率和存活率相互比较差异均无显著统计学意义(P>0.05)。冰冻保存组血小板24 h回收率和存活率均低于其他各组(P<0.05)。结论改良的PAS-ⅢM能够替代血浆在低温条件下用于血小板的保存,能维持血小板的体内功能。     

Stability of platelet surface antigens during storage

We measured changes in A, B, 2H, PlA1, and HLA Class I antigens on human platelets stored as routine platelet concentrates (PCs) in 50 to 60 ml of citrate-phosphate-dextrose-adenine (CPDA-1) plasma in polyolefin (PL 732) bags at 22 degrees C with continuous cartwheel rotation. Samples were obtained at 1, 3, 5, and 10 days of storage; incubated with human IgG anti-A, -B, -HLA and -PlA1; incubated with mouse monoclonal 125I-labeled anti-human IgG; centrifuged through phthalate ester oils; and assayed in a gamma scintillation counter. Additionally, group O platelets were analyzed using 125I-labeled IgM mouse monoclonal anti-Type 2H. Mean values for molecules of Ig bound per platelet showed that platelet surface antigens A, B, 2H, PlA1, and HLA Class I showed no significant change during 10-day storage as routine PCs in CPD-A1 in PL 732 bags. Identical radioassays were performed with platelets incubated at 22 degrees C in plastic test tubes for 24 hours in homologous plasma from donors negative for the respective antigens and in a variety of artificial media with albumin and lipids. No significant changes occurred in any of the surface antigens, except for the loss of approximately 50 percent of the blood group A antigen from platelets stored in O plasma or in albumin media. These data indicate that HLA, PlA1, and type 2H structures do not readily dissociate from the platelet membrane during storage, while some blood group A antigens, presumably acquired passively from the plasma, will elute from the platelet under certain conditions. Routine storage conditions are unlikely to alter the immunogenicity of platelets due to a loss of antigen expression.     

Studies of Platelet Concentrates Stored at 22 C and 4 C

As measured by stability of p H, aggregability, and structure, concentrated platelets are better preserved for 72 hours at 4 C than at 22 C. While survival in vivo , as determined with ⁵¹Cr, of platelets stored at room temperature is extremely variable, survival of platelets stored at 4 C is invariably shortened to two to three days. In the treatment of bleeding in thrombocytopenic patients, however, platelets kept at 4 C for 24 to 72 hours are superior to those kept at 22 C with respect to their capacity to shorten the bleeding time and effect hemostasis. The relative ineffectiveness of platelets kept at 22 C for 72 hours appears to be the result of a functional defect which develops during storage at ambient temperature.
We suggest that until a means is found to prevent the functional abnormality which develops during storage at 22 C., platelet concentrates should be refrigerated if they are to be stored for 24 hours or longer prior to being transfused.     

Serotonin uptake at 22 degrees C of stored platelets

In order to ascertain the possibility that platelet serotonin uptake may occur during storage of platelet concentrates (PC) at 22 degrees C with agitation, the high-performance liquid chromatographic procedure was applied to determine serotonin uptake by platelets. Studies at 22 degrees C showed that platelets stored for 4 days exhibited a significant serotonin uptake with a Vmax value of 2.4 X 10(-19) mole/platelet/min and a Km value of 0.62 X 10(-6) M. Incubation of PC with 5 X 10(-6) M serotonin for 1 day at 22 degrees C increased their serotonin contents from 2.2 to 4.2 X 10(-7) mole/10(11) platelets. Thrombin stimulation caused about 80% release of intracellular serotonin from fresh as well as stored platelets, which contained standard serotonin in the same amount as the original amount. These results suggest that a significant serotonin uptake of platelets might occur during in vitro storage at 22 degrees C and stored platelets have retained abilities to sequester extracellular serotonin into dense granules.     

以海藻糖为添加剂冷藏保存血小板悬液的研究

本研究探讨以海藻糖(trehalose)为添加剂的血小板悬液冷藏保存方法。采用核素标记法检测血小板生存时间,用比浊法测定血小板聚集率,诱导剂为终浓度11.2μmol/L的ADP。采集兔心脏血,按常规方法制备浓缩血小板悬液(PCs),在悬液中加入50mg/ml的海藻糖,37℃水浴4小时后,放于4-8℃冰箱保存,冷藏12天后,测定血小板聚集功能和输入自身体内后的生存时间。结果表明:常温和冷藏储存24小时后的PC血小板聚集率分别为(75.3±9.8)%和(80.5±12.5)%;输入兔体内24、48和72小时时的血小板存活率分别为(78.1±7.9)%、(65.4±6.7)%、(57.5±7.2)%和(5.1±2.5)%、(2.8±2.0)%、(0.9±0.8)%。加入海藻糖的PC冷藏保存12天后,血小板聚集率为(77.8±9.5)%;输入体内24、48和72小时时的血小板存活率分别为(75.7±11.0)%、(67.0±8.5)%、(56.8±8.0)%,与常温保存24小时对照相比,两者相近,P值均>0.05。结论:海藻糖能保护冷藏储存的兔血小板,延长冷藏血小板在体内的生存时间,经海藻糖冷藏储存的兔血小板功能完好。     

Loss of high-affinity thrombin receptors during platelet concentrate storage impairs the reactivity of platelets to thrombin

BACKGROUND: The storage of platelet concentrates (PCs) induces a reduction in the platelet surface expression of glycoprotein (GP) Ib alpha. The location of the platelets' high-affinity binding site for thrombin has been postulated as being located on GPIb alpha. This study attempts to determine whether loss or alteration of GPIb alpha during storage of PCs is related to impairment in the reactivity of platelets to thrombin. STUDY DESIGN AND METHODS: In this study, platelet surface expression of GPIb alpha was monitored by means of flow cytometry, throughout standard storage of PCs for up to 10 days. Two thrombin- induced platelet responses, the binding of radiolabeled fibrinogen and the platelet surface expression of P-selectin, were evaluated. Thrombin- binding assays were also performed to assess the number of thrombin receptors in platelets. RESULTS: The surface expression of the GPIb/IX complex declines during storage of PCs. The thrombin-induced maximal binding of fibrinogen in platelets stored for 3, 7, and 10 days was 77 +/? 7 percent, 60 +/? 20 percent, and 34 +/? 25 percent, respectively, of that found in fresh platelets. Moreover, the concentration of thrombin needed for 50 percent of platelets to express the CD62 antigen P-selectin at the surface increased from 0.05 U per mL in fresh platelets to 0.11, 0.56, and 1.2 U per mL in platelets stored for 3, 7, and 10 days, respectively. Thrombin-binding experiments demonstrated a significant reduction in the number of high-affinity binding sites throughout storage of PCs (55 +/? 21 sites/platelet in 10-day-stored platelets vs. 73 +/? 25 in fresh platelets). A significant correlation was also observed between the number of high-affinity thrombin-binding sites and surface expression of GPIb alpha. Selective blockage of the thrombin-binding site on GPIb alpha with monoclonal antibody LJ-Ib10 also inhibited the response of fresh platelets to thrombin, up to a level equivalent to that found in 3-day-stored platelets. CONCLUSION: The loss of the GPIb alpha-located high-affinity thrombin-binding site may impair the ability of platelets to become activated by thrombin as storage time increases.     

Circulation of Concentrated One‐day‐old Platelets in Vivo

Platelet concentrates of rat and human origin were stored at 22 C or at 4 C for up to 24 hours without additives. Transfusion of these concentrates into thrombocytopenic recipients demonstrated that: (1) storage of rat or human platelets at 4 C for up to 24 hours did not affect their recovery in vivo ; (2) storage at 22 C resulted in a marked reduction in the viability of rat platelets but only a slight reduction in the viability of human platelets as adjudged by these criteria; (3) at 24 hours posttransfusion, the residual increment of platelets stored at 22 C was significantly higher than that of platelets stored at 4 C. The pH of the concentrates (rat and human) stored at 4 C remained slightly alkaline while the pH of those stored at 22 C., especially rat platelets, was significantly reduced. The deleterious effects of storage of platelets at 4 C are well known. These effects, however, do not preclude their usefulness when a limited objective of arresting or preventing hemorrhage for short periods is pursued. When daily platelet transfusions are feasible, platelets stored for 24 hours at 4 C in the absence of fresh material are adequate for clinical use.