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.     

Phosphatidylserine exposure in platelet concentrates during the storage period: differences between the platelets collected with different cell separators.

BACKGROUND AND OBJECTIVES: Platelet alterations occur during the production and storage of platelet concentrates, the so called "storage lesion". We studied the platelet alterations during the storage period in apheresis concentrates, employing flow cytometry for phosphatidylserine (PS) detection on platelets during the five days of storage. MATERIAL AND METHODS: Twenty-seven single donor platelet concentrates harvested with the Cobe Trima, Baxter Amicus, or Haemonetics MCS+ were analyzed for PS exposure by flow cytometry on the day of production (day 1) and on days 3 and 5 of storage. Furthermore PS expression was analyzed in platelet donors' blood samples withdrawn before plateletpheresis. RESULTS: PS expression on platelets gave the following median values: in blood donors before apheresis it was 1.12% (0.13-1.78) in platelets concentrates on the first day (2 h after apheresis) 2.06% (0.66-15.2), the third day 6.57% (1.98-51.13) and the fifth day 23.04% (3.86-80.23). All differences between median values of PS expression in blood samples before apheresis, and platelets concentrates on days 1, 3 and 5 of storage, are statistically significant. The expression of PS in platelet concentrates was analyzed in relation to the blood cell separator used for the collection procedure and showed the following results: on day 1 the median values of PS in platelet concentrates collected with the three different blood cell separators, Trima, Cobe and MCS, did not show statistically significant differences. On day 3, the platelets concentrates collected with the Trima and with the MCS showed differences that were statistically significant. Those were respectively 10.59% (4.56-51.13) and 3.53% (1.98-12.61), p = 0.005. The PS expression in platelet concentrates collected with the Trima and MCS showed differences that are also statistically significant on day 5 at respectively 32.4% (9.61-80.23) and 8.57% (3.86-48.42), p = 0.005. CONCLUSIONS: PS exposure in platelet concentrates on days 3 and 5 rise to levels that could compromise the quality of the platelet units. Improvements in standardized platelet quality controls, and in platelet collection systems are required to reduce the storage lesions in platelets concentrates.     

流式细胞术测定保存血小板再表达CD62p方法的建立及应用

目的　①建立并优化测定血小板再表达膜表面激活标志物CD6 2p能力的流式细胞术 (FCM) ;②测定2 2℃保存液体血小板不同保存期再表达CD6 2p的能力 ,以评价该指标在血小板质量监测以及新的保存方法研究中的应用价值。方法　①采用浓度梯度法优化GPRP浓度条件 ,采用析因设计优化凝血酶浓度和 37℃孵育时间条件 ,寻找最佳阴、阳性对照 ;②将 13份血小板按AABB标准保存 ,并延长保存至 12d ,测定每天血小板CD6 2p再表达率 ,与其相应体内存活期作直线相关性分析。结果　①约 1.5× 10 6个血小板内加入 2 .5mmol/LGPRP可有效防止过量凝血酶诱导的聚集反应 ,孵育条件以 1U/L凝血酶 37℃ 15min然后室温置 15min最佳。采用新鲜富含血小板血浆(FPRP)为阴性对照 ,以凝血酶激活FPRP为阳性对照效果良好。② 2 2℃保存血小板CD6 2p再表达率与其相应体内存活期呈明显正相关 ,单份相关系数 (r) 0 .91～ 0 .99,总体相关系数为 0 .99。结论　①优化建立了一种灵敏简便 ,稳定性和重复性并好的流式细胞术方法用于测定保存血小板CD6 2p再表达率 ;②保存液体血小板CD6 2p再表达率与其相应体内存活期呈明显正相关 ,且相关性良好 ,提示CD6 2p再表达率是监测保存血小板质量的良好指标 ,同时还可能在新的保存方法研究和临床血小板功?     

Annexin V and platelet antigen expression is not altered during storage of platelet concentrates obtained with the AMICUS cell separator.

During storage of platelet concentrate the so-called "storage lesion" occurs. During this time, platelets loose their morphological and functional capacities that are necessary for proper in vivo efficacy following transfusion. Annexin V represents a marker for apoptosis. In this study, Annexin V and additional antigens were analyzed by flow cytometry. Platelet concentrates were obtained with a new cell separator (AMICUS Separator, Fenwal). Following apheresis, platelet units were stored for an experimentally prolonged time of seven days. Daily aliquots of the platelet-rich plasma were obtained to measure Annexin V and platelet antigens CD62p, CD63, CD41a, CD42b, and the binding of fibrinogen. All analyses were performed using flow cytometry. During storage, no significant changes in mean channel fluorescence intensity (MCFI) of CD41a (P = 0.99) and CD42b (P = 0.29), percentage of CD62p+ and CD63+ platelets (P = 0.23 for CD62p; P = 0.52 for CD63), and the binding of fibrinogen to platelets occurred (P = 0.85). Also, the expression of Annexin V remained constant with no significant change (P = 0.36). This study shows that antigens of platelets, obtained with the AMICUS cell separator are well preserved during storage. Regarding Annexin V, no obvious signs of apoptosis can be detected by flow cytometry. These findings demonstrate the high degree of biocompatibility of the apheresis device and storage container.     

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.     

Platelet function testing during 5-day storage of single and random donor plateletpheresis.

Platelet concentrates are routinely manufactured from whole blood by differential centrifugation (random donor platelets-RDP) or by plateletpheresis (single donor platelets-SDP). These platelet concentrates have a storage period of 5 days and many different approaches exist to measure the condition of platelets during their storage. In this study, platelet aggregation testing using adenosine diphosphate (ADP) and collagen and flow cytometric platelet activation analysis using CD41 FITC and CD62 PE before and after ADP was performed on days 1, 3 and 5 of storage of platelet preparations. Thirty three RDPs, stored in Baxter and Kansuk blood bags and 18 SDPs stored in Fresenius blood bags were evaluated. In RDPs and in SDPs; ADP and collagen induced PA responses were decreased significantly on the 3rd and 5th days compared to 1st day. CD62 positive platelet percentage after ADP were decreased significantly on the 3rd and 5th days compared to the 1st day in Kansuk((R)) bags. Flow cytometric analysis revealed minor changes in CD41 expression after ADP on the 3rd day compared to 1st day and on the 5th day compared to 3rd day. Differences in CD62 positive platelet percentage were not significant between the RDPs and SDPs. Our results suggest that: (1) ADP and collagen induced PA responses decrease both in RDPs and SDPs during storage. (2) Flow cytometric analysis does not show major significant changes in platelet activation after ADP during storage. (3) Continous shaking on the agitator does not cause a significant change in CD62 positive platelet percentage during storage. (4) Platelet aggregation responses in RDPs stored in Baxter((R)) and Kansuk((R)) blood bags do not differ during storage.     

Measurement of phosphatidylserine exposure during storage of platelet concentrates using the novel probe lactadherin: a comparison study with annexin V

BACKGROUND: Annexin V binding to platelets (PLTs) is considered the gold standard for monitoring phosphatidylserine (PS) exposure. However, recent comparison of annexin V with the new calcium-independent PS probe lactadherin revealed that annexin V requires a certain threshold of PS exposure (2%-8%) for binding to occur. The aim of this study was to compare annexin V and lactadherin labeling of PLTs in PLT concentrates (PCs).
STUDY DESIGN AND METHODS: Optimal labeling conditions for lactadherin and annexin V were established and then compared in either resting or calcium ionophore (CI)-activated PLTs from normal whole blood. Furthermore, 40 PCs (20 apheresis-derived and 20 pooled buffy coat–derived) were stored under standard blood bank conditions and PLT activation was monitored by measuring PS exposure with annexin V and lactadherin along with CD42b, CD61, and CD62P by flow cytometry on Days 1, 3, 5, and 7.
RESULTS: Lactadherin reported a higher exposure of PS than did annexin V in normal PLTs at submaximal doses of CI. PLTs from both types of concentrate, as expected, demonstrated evidence of increased activation during storage using annexin V, lactadherin, CD42b, or CD62P. However, a significantly higher percentage of PS-positive PLTs was found with lactadherin than annexin V.
CONCLUSION: PS exposure on the surface of stored PLTs has been previously underestimated due to the wide use of annexin V. Lactadherin provides a truer reflection of the degree of PS exposure and offers a new calcium-independent approach to studying PLT activation and/or apoptosis.     

Role of mitochondria in the maintenance of platelet function during in vitro storage

Aims/Objectives: Platelets undergo structural and biochemical alterations during in vitro storage and these are collectively called platelet storage lesions (PSL). The mitochondrion is an important cell organelle involved not only in energy production but also in the regulation of cellular functions and viability. This implies that some platelet functions may be regulated by mitochondria; hence, preservation of mitochondrial functions may be important for the maintenance of platelet quality in stored platelet concentrates (PCs). This work describes the effects of various compounds on mitochondrial functions important for the maintenance of platelet quality in in vitro stored PCs. Methods: PCs were stored at 22 °C with gentle agitation in the presence or absence of 2,4‐dinitrophenol, antimycin A, acetyl‐l ‐carnitine and ascorbic acid. The effects of these products on platelet quality were assessed by analysing glucose and lactate concentrations, pH of the storage medium, shape of the platelets, mitochondrial membrane potential and depolarisation, surface expression of CD62P and collagen‐induced platelet aggregation. Results: 2,4‐Dinitrophenol and antimycin A increased PSL levels, whereas acetyl‐l ‐carnitine reduced the level of changes in pH and mitochondrial depolarisation. Ascorbic acid in the storage medium resulted in improved levels of collagen‐induced platelet aggregation. However, none of the examined reagents suppressed CD62P expression in platelets. Conclusions: These results suggest that preservation of mitochondrial function is fundamental, but not fully sufficient, for the maintenance of platelet in vitro quality during storage. Further research is necessary to develop methods for preserving both mitochondrial and platelet functions in in vitro stored PCs.     

糖基化修饰血小板冷藏稳定性研究

本研究旨在观察尿苷二磷酸半乳糖（UDP-6ai）糖基化修饰人血小板的稳定性、理化指标及体外功能变化。实验分为室温对照组、冷藏对照组以及修饰组（U＋4组和4＋U组）。机采人浓缩血小板悬液，4℃保存前或后添加适量UDP—Gal，进行糖基化修饰，分别于0、1、3、5、7、14天，通过荧光标记识别血小板膜糖蛋白末端半乳糖残基的凝集素（FITC—RCAⅠ）检测糖基化修饰效果；pH计检测血小板悬液pH值；血细胞计数仪检测血小板平均体积；比浊法测定血小板聚集率；流式细胞术检测血小板活化标志CD62P、血小板膜蛋白CD42b及Ps的表达。结果表明：保存14天时，修饰组血小板RCAⅠ结合率是室温组的5—6倍；修饰组血小板悬液pH低于室温组，但二者之间无显著性差异（P〉0．05）；保存14天时室温组和修饰组血小板平均体积分别为10．6±1．9fL和11．14±1．1fL，二者之间无显著性差异（p〉0．05）；各组体外聚集活性在保存过程中均逐渐下降，但修饰组优于室温组（p〈0．05）；流式检测结果显示，保存1—5天修饰组CD62P、CD42b和Ps表达的阳性率与新鲜血小板无显著性差异（P〉0．05），但保存14天时，CD62P和Ps表达的阳性率升高，CD42b表达的阳性率降低，与新鲜血小板相比差异极显著（p〈0．01）。结论：在修饰血小板保存过程中，糖基化修饰的效果稳定，修饰血小板的pH和平均体积均处于正常值范围之内，聚集活性良好，优于室温保存组，但在保存5天后表现出不同程度的活化。     

The effect of storage on the expression of platelet membrane phosphatidylserine and the subsequent impacton the coagulant function of stored platelets

BACKGROUND: Platelet concentrates (PCs) derived from whole blood and stored under standard blood bank conditions undergo changes that are referred to as the platelet storage lesion. This study assesses the effect of PC preparation and storage on the distribution of phosphatidylserine (PS) in the platelet membrane and the effect that this distribution may have on the thrombogenic potential of stored PCs. STUDY DESIGN AND METHODS: Fresh platelets and PCs donated by healthy donors were obtained. PCs derived from platelet-rich plasma were studied on Day 1, Day 3, and Day 6 of storage under blood bank conditions. RESULTS: Platelet aggregation after exposure to the platelet agonists ADP and epinephrine singly declined progressively, but, when ADP and epinephrine in combination and collagen and thrombin in combination were used as agonists, the decline in platelet aggregation was less marked. PS expression as measured by Annexin V binding (mean and SD) was 2.02 +/- 0.93 percent in fresh platelet samples and increased to 5.39 +/- 4.2 percent on Day 1, 22. 1 +/- 7.1 percent on Day 3, and 39.5 +/- 12.1 percent on Day 6. Platelet prothrombinase activity (mean +/- SD) as measured by thrombin generation increased from 1.49 +/- 0.7 micro per mL in fresh platelet samples to 3.68 +/- 1.1 micro per mL in Day 1 platelets (p<0.001), 5.15 +/- 2.5 micro per mL in Day 3 platelets (p<0.001), and 4.65 +/- 2.48 micro per mL in Day 6 platelets (p<0. 001). CONCLUSION: These results show that PS expression increases after preparation of PCs from platelet-rich plasma and rises progressively during platelet storage under blood bank conditions. Furthermore, the greater PS expression is associated with increased platelet- dependent thrombin-generating capacity.     

Thrombin-triggered platelet apoptosis

BACKGROUND: Thrombin is primarily known as a coagulation factor and as an inducer of platelet activation and aggregation. It has been reported that thrombin modulates apoptosis of nucleated cells. OBJECTIVES: The current study investigated whether thrombin can affect apoptosis in anucleated human platelets. METHODS: Using flow cytometry, we studied platelet apoptosis at the single-cell level, analyzing markers of mitochondrial and cytoplasmic apoptosis. Western blotting was also employed, in addition to flow cytometry, for determining the expression of Bcl-2 family proteins. RESULTS: We found that human alpha-thrombin induced four key manifestations of apoptosis in human platelets: (i) mitochondrial inner transmembrane potential (DeltaPsi m) depolarization; (ii) strong expression of pro-apoptotic Bax and Bak proteins but only weak expression of anti-apoptotic Bcl-2 protein; (iii) caspase-3 activation; and (iv) phosphatidylserine (PS) exposure. CONCLUSIONS: This study demonstrates that, aside from its 'classical' function as an inducer of platelet activation, thrombin can trigger platelet apoptosis, where it acts as a death ligand. These data indicate that thrombin triggers platelet apoptosis by impacting on several intracellular apoptotic targets, including shifting the balance between Bcl-2 regulatory proteins in a pro-apoptotic direction, depolarizing the inner mitochondrial membrane, activating the executioner caspase-3, and stimulating aberrant exposure of PS on the platelet surface.     

不同方法制备的血小板在保存期内活化状态研究

目的研究白膜回浆法（Bc）制备的浓缩血小板和血细胞分离机（Trima）制备的单采血小板在保存期内的活化状态。方法采用流式细胞术和酶联免疫吸附法分别检测BC和Trima制备的血小板在5d保存期内血小板表面CD62P和血浆可溶性cD62P（scD62P）的表达。结果在O～5d的保存期内,两种方法制备的血小板表面CD62P和血浆sCD62P的表达均随保存时间推移而增加;BC制备的浓缩血小板CD62P阳性率和血浆sCD62P的表达均明显高于Trima制备的单采血小板。结论BC制备的浓缩血小板活化程度高于Trima制备的单采血小板,其制备工艺需改进,以减少血小板活化水平。     

Binding of activated platelets to WBCs in vivo after transfusion

BACKGROUND: During preparation and storage of apheresis concentrates, platelets are being activated. One of the alterations that occur during this process is an increased expression of P-selectin (CD62p) on the cytoplasmic surface of platelets. This neoepitope represents a ligand for the binding of platelets to WBCs. It has been suggested that the activation of platelets is associated with the sequestration of platelets after transfusion. In this in vivo study, the binding of platelets to WBCs was analyzed following transfusion of platelet concentrates (PCs). STUDY DESIGN AND METHODS: Double apheresis concentrates were prepared with two different cell separators. One of the split products was stored for 1 to 2 days and the other one for 3 to 5 days. Flow cytometry was applied to analyze the degree of platelet activation in vitro, and also to measure the extent of platelet binding to WBC subclasses in vivo after transfusion into patients. RESULTS: The results of this study show that platelet activation occurs during apheresis and storage of PCs. After transfusion of the PCs, no significant binding of platelets to T or B-cells could be detected. However, a significant binding of platelets to monocytes and neutrophil granulocytes occurs. While in Baxter PCs stored for 1-2 days the amount of platelet-leukocyte aggregates in vivo was higher compared to COBE PCs, no such difference could be detected anymore for the PCs stored for 3-5 days. CONCLUSION: This study demonstrates that binding of activated platelets occurs to monocytes and neutrophil granulocytes but not to T- and B-cells in the circulation after transfusion. In addition, the interaction of platelets and WBCs is dependent on the degree of P-selectin expression. Platelets showing a higher degree of activation adhere to WBCs to a higher degree than nonactivated platelets.     

The influence of irradiation on stored platelets

Platelet concentrates intended for transfusion to immunosuppressed patients are irradiated to minimize transfusion-induced graft-versus-host disease. Because few reports describe how irradiation influences stored platelets, the authors studied whether 5000 rad of gamma irradiation, the maximum dose currently used clinically, altered platelets in vitro. Platelet concentrates were stored for either 1 day or 5 days in plastic (PL 732) containers before gamma irradiation. One unit of a pair of identical platelet concentrates was irradiated; the second unit served as a control. Irradiation did not alter platelet morphology, mean platelet volume, expression of platelet-factor-3 activity, response to hypotonic stress, extent of discharge of lactate dehydrogenase, release of beta-thromboglobulin, formation of thromboxane B2, nor the ability to undergo synergistic aggregation. The lack of any substantial change was observed whether the platelet concentrates were stored initially for either 1 day or 5 days. These results suggest that stored platelets are not altered deleteriously by irradiation with 5000 rad.     

The combined effect of platelet storage media and intercept pathogen reduction technology on platelet activation/activability and cellular apoptosis/necrosis: Lisbon-RBS experience.

Platelets are known to undergo shape change, activation, a release reaction and apoptosis/necrosis during processing and storage, all of which are collectively known as the platelet storage lesion. Any additional processing may have some deleterious impact on platelet activability and functional integrity, which need to be investigated. This preliminary investigation was undertaken to establish the combined effects of standard platelet storage media and the intercept pathogen reduction technology on platelet activation and activability during 7 day storage, using buffy-coat derived platelets in standard storage media containing 35% plasma (N=24). P-selectin (CD62p) expression, a classical marker of platelet activation, and phosphatidylserine (PS) exposure on the platelet surface membrane, a hallmark of cellular necrosis/apoptosis, were both measured by flow cytometry. The results reveal significant increases in activation, from an average of 22.7% on day 1 before treatment to 31.6% on day 2 after treatment and 58.7% at the end of storage. Concomitantly, the basal expression of PS was slightly increased from 1.9% to 2.8% at day 2 after treatment and 7.3% at the end of storage. However, the functional reserve of platelets during storage, which reflects their capability to undergo activation and the release reaction when platelets were challenged with either calcium ionophore or thrombin, was relatively well maintained. These preliminary data confirm the earlier data on the use of intercept, and for the first time, based on the assessment of platelet functional integrity, suggest that platelet functional reserve is relatively well maintained, with little change in the formation of apoptotic cells.     

Platelet concentrates stored for 5 days in a reduced volume of plasma maintain hemostatic function and viability

BACKGROUND: Platelet concentrates prepared from whole blood are generally suspended in a standard volume of 50 to 60 mL of plasma and can be stored thus at 20 to 24 degrees C for up to 5 days. In vitro studies suggested that this plasma volume could be reduced to 30 to 35 mL without impairing platelet function. STUDY DESIGN AND METHODS: This study evaluated whether platelets stored for 5 days in a reduced volume (30-35 mL) of plasma maintained their in vivo viability, hemostatic function, and recovery in recipients. Paired autologous platelet survival studies were done in 20 adult volunteers to assess platelet viability. A rabbit ear bleeding-time model was used to compare the hemostatic effectiveness of human platelet concentrates stored for 5 days in the standard or reduced volume of plasma. Platelet recovery was compared in thrombocytopenic hospital patients. RESULTS: Paired platelet survival studies indicated no significant difference between the values in platelet concentrates stored for 5 days in the reduced volume of plasma and the values in those stored in the standard volume. In the animal model, there was no significant difference in the bleeding times achieved by either set of platelet concentrates. The platelet count increments in thrombocytopenic patients were measured. The platelet count increments in patients who received reduced-volume platelet concentrates were as good as the increments achieved in patients given standard-volume concentrates. CONCLUSION: The in vivo viability, recovery, and hemostatic function of platelets collected in polyvinylchloride plastic containers and stored in 30 to 35 mL of plasma for 5 days are maintained as well as those of platelets stored in 50 to 60 mL of plasma.     

Procoagulant surface exposure and apoptosis in rabbit platelets: association with shortened survival and steady-state senescence.

BACKGROUND: The signal(s) for removal of senescent platelets from the circulation are not fully understood; phosphatidylserine (PS) expression on platelets and another marker of apoptosis, loss of mitochondrial inner membrane potential (DeltaPsim), have been implicated in platelet clearance. OBJECTIVE: To investigate whether shortened platelet survival and steady-state platelet senescence are associated with increased surface exposure of PS and DeltaPsim collapse. METHODS: Survival of in-vitro biotinylated rabbit platelets treated with thrombin or Ca(2+)-ionophore A23187 was tracked by flow cytometry after injection. Steady-state platelet senescence was investigated by infusing biotin to label a platelet cohort. PS expression and DeltaPsim of in-vitro biotinylated platelets and of the aging platelet cohort biotinylated in-vivo were measured by flow cytometry using annexin V-FLUOS and the DeltaPsim-sensitive dye CMXRos, respectively. RESULTS: Although PS expression, DeltaPsim and survival of thrombin-degranulated platelets were similar to those of control platelets, increasing concentrations of A23187 caused increased surface exposure of PS and progressive shortening of platelet survival; only one-sixth of PS-expressing platelets also exhibited DeltaPsim loss. The cohort of senescent, biotinylated platelets remaining in the circulation at 96 h had increased exposure of PS and collapsed DeltaPsim; of the 17% of PS-expressing platelets, one-third did not exhibit DeltaPsim loss. There was also an increase in platelets with collapsed DeltaPsim but not expressing PS. CONCLUSIONS: Platelets with shortened survival and senescent platelets have increased surface exposure of PS, that may be involved in their clearance. PS expression can occur independently of DeltaPsim collapse and conversely, in aged platelets, DeltaPsim loss can occur independently of PS expression.     

Quantitation of CD62, soluble CD62, and lysosome-associated membrane proteins 1 and 2 for evaluation of the quality of stored platelet concentrates

BACKGROUND: Platelets become activated during storage, which results in secretion of granules, vesiculation of microparticles, secretion of protein, and a number of other biochemical and morphologic processes that decrease the utility of platelet concentrates stored for transfusion. STUDY DESIGN AND METHODS: To evaluate the quality of stored platelet concentrates, the cell surface expression of specific activation-dependent antigens (CD62 and lysosome-associated membrane proteins 1 and 2 [LAMP-1, LAMP-2]) on platelets stored in a hospital blood bank over a 7-day period was examined. Relative microparticle counts and the expression of CD62 by microparticles, as well as platelet concentrate supernatant levels of soluble CD62, were determined. RESULTS: The percentage of platelets expressing CD62 increased significantly from Day 1 to Day 5 (p < 0.05) of storage; the mean fluorescence values for CD62 did not. In contrast, the mean fluorescence values of LAMP-1 and LAMP-2 rose significantly (p < 0.01 and p < 0.05, respectively) between Days 1 and 5. Significant declines in CD62, LAMP-1, and LAMP-2 percent expression and mean fluorescence were seen on Day 6 of storage (p < 0.001). Microparticle numbers increased significantly during storage and correlated with levels of CD62 protein (free and membrane-bound) (r = 0.95 vs. Day 2, p < 0.05; r = 0.88 vs. Day 5, p < 0.05). CONCLUSION: Flow cytometric evaluations of the expression of cell surface CD62, LAMP-1, and LAMP-2 are complementary tests that, especially when used in conjunction with the quantitation of CD62 protein, provided a simple and effective means of evaluating the quality of platelet concentrates stored for transfusion.     

冰冻保存血小板新亚群及其新特性体外研究

目的　探讨血小板冰冻保存后血小板新亚群的产生和其获得新特性的分子基础。方法　采用流式细胞术在不同条件下比较分析新鲜血小板和冰冻保存血小板膜表面功能分子表达变化差异。结果　根据表达和再表达CD6 2p能力不同可将冰冻血小板分为三个亚群 ,CD6 2 p再表达率为 5 1 .7%± 7.8% ;新鲜血小板只有一个明显的亚群 ,CD6 2 p再表达率为 98.3%± 0 .6 %。结合磷脂酰丝氨酸 (PS)、CD6 2 p和CD4 2b分子变化可将冰冻血小板分出新的亚群 ,其显著特征包括PS阳性和CD4 2b分子密度显著降低 ;而CD6 2p阴性亚群对钙离子的激活更为敏感。结论　血小板通过冰冻保存后 ,产生了新亚群。新亚群膜表面功能分子的变化及其表达能力改变可能与其体内存活率减低而即刻止血功能增强密切相关     

Usefulness of delta value of platelet parameters on ADVIA 120 for the functional reactivity of stored platelets

An agonist‐induced expression of CD62P by flow cytometry analysis for evaluating platelet functional reactivity has some disadvantages. We investigated the usefulness of platelet parameters by ADVIA 120 to predict an agonist‐induced expression of CD62P in stored platelets. The CD62P expression by flow cytometry and the platelet parameters by ADVIA 120 were studied in samples from 27 platelet pheresis products. Delta (Δ) values were calculated as the degree of change of the platelet parameters studied with or without adenosine 5′‐diphosphate sodium (ADP) stimulation. The CD62P expression of the ADP‐activated platelets were correlated with the Δplatelet count (r=0.517) in the short‐term storage group (within 10 hr from preparation), with the platelet component distribution width (PCDW) without ADP (r=?0.744) and the ΔPCDW (r=?0.755) in the long‐term storage group (after 10 hr from preparation). Therefore, the delta values of platelet parameters on ADVIA 120 analysis in platelets between with and without ADP stimulation could be useful as a simple predictor for the functional reactivity of stored platelets. J. Clin. Lab. Anal. 24:38–43, 2010. © 2010 Wiley‐Liss, Inc.