Cold-stored platelets: A product with function optimized for hemorrhage control

Early administration of blood products following severe trauma is pivotal for establishing hemostasis and achieving successful outcomes. Platelet transfusions, in particular, provide rapid control of hemostasis and help to restore platelet dysfunction induced by trauma. In the U.S. platelets used for therapeutic purposes are stored at room temperature with a limited shelf life of 5-7 days. Issues with room temperature storage of platelets, including an increased risk of bacterial growth and a decline in platelet hemostatic function, have led to a resurgence in interest in cold-stored platelets for therapeutic transfusion. This review presents the current state of cold-stored platelets and cold-stored whole blood as treatment for actively bleeding patients. Usage of cold stored platelets in alternative areas, such as in the field of regenerative medicine, is also discussed.     

网织血小板和血小板参数在急性冠脉综合征中的变化及意义

目的:探讨急性冠脉综合征(ACS)患者网织血小板(RP)和血小板参数的变化及意义。方法:随机选取ST段抬高心肌梗死(STEMI)27例为STEMI组,非ST段抬高心肌梗死(NSTEMI)25例为NSTEMI组,不稳定心绞痛(UA)31例为UA组,健康体检者30例为对照组进行检测。用CD61-PE单抗标记血小板,噻唑橙(TO)为染料,应用富含血小板血浆(PRP)法检测RP;用COULTERJT型全自动血液分析仪测定血小板参数[血小板计数(PLT)和血小板平均体积(MPV)]。结果:STEMI、NSTEMI、UA组3组RP%均显著性高于对照组(P<0.05);STEMI、NSTEMI组的RP%显著高于UA组(P<0.05);STEMI、NSTEMI组的MPV均显著高于对照组(P<0.05)。结论:RP可能成为评估ASC患者血小板活性的新指标,它和MPV的检测可能对ACS有着一定的临床意义。     

Quality of irradiated and non-irradiated plateletpheresis concentrates stored in platelet additive solution

Introduction

Platelet additive solutions (PAS) allow to maintain platelet storage properties in platelet concentrates (PCs). The aim of the present study was to evaluate the in-vitro quality of irradiated and non-irradiated PCs, suspended in PAS, over a storage period of 6 days.

The phospholipid composition and cholesterol content of platelet-derived microparticles: a comparison with platelet membrane fractions

BACKGROUND: The processes that govern the distribution of molecules between platelets and the microparticles (MP) they release are unknown. Certain proteins are sorted selectively into MP, but lipid sorting has not been studied. OBJECTIVES: To compare the phospholipid composition and cholesterol content of platelet-derived MP obtained with various stimuli with that of isolated platelet membrane fractions. METHODS: Washed platelets from venous blood of healthy individuals (n = 6) were stimulated with collagen, thrombin, collagen plus thrombin, or A23187. Platelet activation, MP release and antigen exposure were assessed by flow cytometry. MPs were isolated by differential centrifugation. Platelet plasma-, granule- and intracellular membranes were isolated from platelet concentrates (n = 3; 10 donors each) by pressure homogenization and Percoll density gradient fractionation. The phospholipid composition and cholesterol content of MPs and membrane fractions were analyzed by high performance thin layer chromatography. RESULTS: The phospholipid composition of MPs was intermediate compared with that of platelet plasma- and granule membranes, and differed significantly from that of intracellular membranes. There were small but significant differences in phospholipid composition between the MPs produced by the various agonists, which paralleled differences in P-selectin exposure in case of the physiological agonists collagen, thrombin, or collagen plus thrombin. The cholesterol content of MPs tended to be higher than that of the three-platelet membrane fractions. CONCLUSIONS: Regarding its phospholipid content, the MP membrane is a composite of the platelet plasma- and granule membranes, showing subtle differences depending on the platelet agonist. The higher cholesterol content of MPs suggests their enrichment in lipid rafts.     

Alzheimer and platelets: Low-density platelet populations reveal increased serotonin content in Alzheimer type dementia

Introduction

Alzheimer's disease (AD) is a progressive form of dementia characterized by an increase in the toxic substance β-amyloid in the brain. Platelets display a substantial heterogeneity with respect to density. They further contain a substantial amount of β-amyloid precursor protein. Platelets take up and store serotonin (5-HT) that plays an important role in the pathogenesis of severe depression. The current study aims to investigate platelet serotonin content in different platelet density populations.

Material and methods

The study involved 8 patients (age 70 ± 8 (SD) years) (3 females/5 males) with moderate AD. 6 healthy elderly subjects (age 66 ± 9 (SD) years) (3 females/3 males) served as controls. The platelet population was divided into 17 subpopulations according to density, using a linear Percoll™ gradient. Platelets were counted in all fractions. After cell lysis an ELISA technique was employed to determine the 5-HT content in each platelet subfraction.

Results

The two study groups did not differ significantly regarding platelet distribution in the gradients, but AD sufferers have a significantly higher 5-HT content (p < 0.05) in the lighter platelet populations.

Discussion

AD-type dementia proved to be associated with lighter platelets containing more 5-HT. It is possible that platelets from AD patients release less 5-HT. It is speculated that AD synapses are affected in a manner comparable to platelets, which could explain why 5-HT reuptake inhibitors are less effective in AD dementia.     

急性心肌梗死患者血小板活化标志物与超微结构的变化

目的 探讨急性ST段抬高心肌梗死(STEMI)患者血小板α颗粒糖蛋白(platelet activationdependent granule membrane protein,PADGEM or GMP-140,CD62P)、血小板源生长因子受体αβ亚型(platelet-derived growth factor receptor-αβ,PDGFR-αβ)表达,血小板超微结构的变化.方法 采用流式细胞仪测定36例急性ST段抬高心肌梗死患者和同期34名健康体检者(对照组)外周血中血小板的血小板a颗粒糖蛋白(CD62P)、血小板源生长因子受体αβ亚型(PDGFR-αβ)表达,采用扫描电子显微镜观察血小板超微结构,并进行比较.结果 急性STEMI组治疗前血小板α颗粒糖蛋白、血小板源生长因子受体αβ表达率分别为(3.65±1.87)%,(0.43±0.39)%,治疗后为(0.96±0.79)%,(0.28±0.24)%,对照组相应为(0.67±0.35)%,(0.27±0.22)%.STEMI组的血小板α颗粒糖蛋白、血小板源生长因子受体αβ表达率,治疗前较对照组高(分别为P＜0.01和P＜0.05),治疗后明显降低.STEMI组扫描电镜观察血小板表而超微结构变化明显.结论 STEMI血小板处于活化状态,血小板活化指标血小板α颗粒糖蛋白可作为冠心病病情监测的有效指标,血小板源生长因子受体αβ有参考价值,扫描电镜观察可发现血小板活化的超微病理结构改变.

Abstract：

Objective To investigate the expressions of platelet activation-dependent granule membrane protein and platelet-derived growth factor receptor-αB, and the ultra-microstructure changes of platelets in patients with acute ST-segment elevation myocardial infarction(STEMI). Method The expressions of platelet activationdependent granule of glycoprotein (CD62P)and platelet derived growth factor receptor αβ subtype (PDGFR-αβ)of platelets in peripheral blood in 36 patients with acute ST-segment elevation myocardial infarction(STEMI) hospitalized and another 34 healthy subjects over the same period (control group) were investigated by flow cytometry and data were analyzed. The changes of ultra microstructure and activity of blood platelets in those patients and control group were observed under the scanning electron microscope. Results The expressions of CD62P and PDGFR-αβin patients with STEMI group before treatment were (3.65 ± 1.87) % and (0.43 ± 0.39) %, respectively, and those after treatment were (0.96 ± 0.79) % and (0.28 ± 0. 24) %, respectively, whereas those in control group were (0.67 ± 0.35) % and (0.27 ± 0.22) %, respectively, which were much lower in control than those in patients with STEMI before treatment (P ＜ 0.01 or P ＜ 0.05) respectively. There were statistically significant differences in the expressions of CD62P and PDGFR-αβ in patients group between pre-treatment and posttreatment (P ＜0.01 or P ＜0.05), respectively. Obvious ultra-microstructure changes of platelet surface in patients with STEMI group were observed. Conclusions Due to platelet activation in AMI, the expressions of CD62P can be used as effective indicators for monitoring coronary heart disease, and the PDGFR-αβ can be used as a reference indicator. The platelet surface ultra-microstructure changes during platelet activation in patients with AMI can be found by scanning electron microscopy.     

Platelet microparticles and soluble P selectin in peripheral artery disease: Relationship to extent of disease and platelet activation markers

Background. There is increased platelet activation in many cardiovascular diseases. This observation may explain the presence of increased levels of platelet microparticles (PMP) in these diseases. However, whether or not levels of PMPs inter‐relate with other markers of platelet activation, such as soluble P‐selectin, or with disease severity, is unknown. We therefore hypothesized raised PMP levels in stable peripheral artery disease (PAD) intermittent claudication (IC), with an additional increase in severe PAD critical limb ischaemia (CLI). Furthermore, we tested the hypothesis that PMP levels are correlated with other markers of platelet activation, such as soluble P‐selectin, membrane bound P‐selectin (CD62P) and CD63.

Methods. Patients with PAD were recruited from the vascular outpatient and inpatient facilities at a teaching hospital. Age‐ and sex‐matched controls were also recruited from healthy volunteers. Venous blood was obtained from 23 patients with severe disease (CLI), 36 with moderate disease (IC), and from 30 healthy controls. The percentage of platelets positive for CD62P and CD63, as well as the numbers of PMPs were defined by flow cytometry. Plasma soluble P selectin was measured by enzyme‐linked immunosorbent assay (ELISA).

Results. PMPs were increased relative to healthy controls in patients with IC, with a further increase in CLI (P<0.001). Soluble P selectin and CD62+ve platelets were raised in both patient groups, but there was no difference amongst the two patient groups. CD63+ve cells were raised only in CLI compared to healthy controls. In multivariate analysis, only PMP and soluble P selectin independently predicted disease severity, and the two markers correlated modestly (r?=?0.345, P<0.001).

Conclusion. Increased PMP and soluble P selectin are both related to the severity of symptomatic PAD. However, it is uncertain if this relationship is a cause or effect of atherosclerosis. This finding may have clinical implications as PMPs have the potential to influence the progression of atheroma as well as promote thrombosis.     

Impaired synthesis and action of antiaggregating cyclic nucleotides in platelets from obese subjects: possible role in platelet hyperactivation in obesity

BACKGROUND: Subjects with central obesity exhibit platelet hyperactivity, which is involved in the atherosclerotic process and therefore can account for the increased risk of cardiovascular morbidity and mortality. The aim of the study was to evaluate whether alterations of platelet function in obesity involve synthesis and/or action of the two antiaggregating cyclic nucleotides adenosine 3',5'-cyclic monophosphate (cAMP) and guanosine 3',5'-cyclic monophosphate (cGMP). MATERIALS AND METHODS: In platelets from 16 obese and 15 control subjects we investigated the influence on platelet responses to the Adenosine-5-diphosphate sodium salt (ADP) exerted by (i) prostacyclin analogue Iloprost (0.31-5 nmol L(-1)) and the cAMP analogue 8-bromo-cAMP (10-500 micro mol L(-1)); and by (ii) nitric oxide (NO) donor sodium nitroprusside (SNP) (5-100 micro mol L(-1)) and the cGMP analogue 8-bromo-cGMP (10-500 micro mol L(-1)). IC(50) (minimal concentration of each inhibitor necessary to reduce platelet response to ADP by half) was determined. Iloprost and SNP ability to increase cyclic nucleotides was also measured. RESULTS: Significantly greater IC(50) were observed in obese subjects than in healthy controls (1.59 +/- 0.16 vs. 0.80 +/- 0.08 nmol L(-1), P = 0.0001 for Iloprost, and 27.6 +/- 6.5 vs. 7.0 +/- 1.7 micro mol L(-1), P = 0.006, for SNP); when data from control and obese subjects were pooled together, IC(50) of Iloprost and SNP correlated with the homeostasis model assessment (HOMA IR), which is a parameter used to measure the insulin resistance (r = 0.588, P = 0.029 and r = 0.640, P = 0.006, respectively). Also the antiaggregating effect of 8-Br-cAMP and 8-Br-cGMP was smaller in the obese subjects. Finally, the ability of Iloprost to increase platelet cAMP and the ability of SNP to increase both cGMP and cAMP were reduced in obese subjects. CONCLUSIONS: Platelet resistance to the antiaggregating effects of prostacyclin and NO in obesity is attributable to impairment of cyclic nucleotide synthesis and action. As cyclic nucleotides are the main effectors of platelet antiaggregation, the resistance to them can account for platelet hyperactivity in obesity.     

Recombinant human thrombopoietin improves platelet counts and reduces platelet transfusion possibility among patients with severe sepsis and thrombocytopenia: A prospective study

Introduction

Thrombocytopenia is prevalent in patients with severe sepsis, and it is associated with mortalities. Effectively adjunctive treatment might be needed to reverse low platelet counts (PCs). With a growing understanding of thrombocytopenia, recombinant human thrombopoietin (rhTPO) is considered a promising beneficial drug. The present study was dedicated to evaluate the efficiency of rhTPO in improving PCs in patients with severe sepsis.

Materials and Methods

We performed a prospective study in patients with severe sepsis between March 2012 and February 2013. All enrolled patients were divided into rhTPO group and control group, depending on whether rhTPO was prescribed or not. Platelet counts and other parameters were measured initially and in the following 15 days.

Results

Totally, 72 patients (38 in the rhTPO group and 34 in the control group) were included. All enrolled parameters exhibited no significant differences between groups at the baseline. Platelet counts showed a significant increase over time in both groups. Faster improvement of PCs in the rhTPO group was observed with a significant difference. Less platelet transfusion occurred in patients who received rhTPO in our study, as well. No drug-related adverse event during the rhTPO therapy was recorded.

Conclusion

The use of rhTPO in combination with conventional medical therapies could significantly improve the PCs in patients with severe sepsis and thrombocytopenia and effectively reduce the platelet transfusion possibility.     

A prospective randomized study of three types of platelet concentrates in patients with haematological malignancy: corrected platelet count increments and frequency of nonhaemolytic febrile transfusion reactions

We prospectively randomized 51 patients with haematological malignancy requiring platelet concentrates (PCs) to receive either single donor plateletpheresis products (SD-PC), PCs made from pooled buffy coats (BC-PC) or pooled units of platelets made by the platelet-rich plasma method (PRP-PC). The leucocyte content of each type of PC was 0.33 (0.03–13.5), 5.68 (0.19–99.0) and 365 (65–910) × 10⁶; median (range), respectively; P  < 0.0001. All red cell transfusions were leucodepleted by filtration. Statistical comparison of the probability of the occurrence of a nonhaemolytic febrile transfusion reaction (NHFTR) following transfusion of PCs in patients in each group showed a significant decrease for the SD-PC and BC-PC groups (0.031 and 0.038, respectively) when compared with PRP-PC (0.171); P  =0.001. The actual corrected platelet count increments (CCI) at 1–6 and 18–24 h post-transfusion for all three types of PC did not differ significantly. We conclude that transfusion of PRP-PC is associated with a significant increase in NHFTR.     

Reactions and platelet increments after transfusion of platelet concentrates in plasma or an additive solution: a prospective, randomized study

BACKGROUND: Reactions after platelet transfusions are rather common and frequently are caused by plasma constituents. In recent developments, the preparation and storage of platelet concentrates (PCs) in a platelet additive solution (PAS-2) have been shown to result in acceptable storage conditions. A major drawback of the use of these PCs is the progressive increase of P-selectin-positive platelets during storage. The clinical benefit of transfusions of PCs in PAS-2 was studied. STUDY DESIGN AND METHODS: PCs prepared from buffy coats were suspended in either plasma or PAS-2 and stored for up to 5 days. Clinical responses were evaluated in a prospective study in 21 patients treated with intensive chemotherapy for hematologic malignancies. Eligible patients were randomly assigned to receive prophylactic transfusions of PCs prepared in either plasma or PAS-2. Reactions and CCIs were recorded after each transfusion. RESULTS: The incidence of reactions in 12 patients given PCs in plasma (n = 192) was 12 percent. Transfusions to 9 patients of PCs in PAS-2 (n = 132) showed a reduction in the incidence of reactions to 5.3 percent (p<0.05). The average 1-hour and 20-hour CCIs after transfusion of PCs in plasma were 20.7 +/- 8. 5 and 11.5 +/- 8.0, respectively. CCIs after transfusion of PCs in PAS-2 were significantly lower: the average 1-hour CCI was 17.1 +/- 6.6 (p<0.001) and the average 20-hour CCI was 9.5 +/- 7.0 (p<0.05). Storage conditions of PCs were optimal: in each group, average 1-hour CCIs of both fresh and stored PCs were similar. The 20-hour CCIs after the transfusion of fresh and stored PCs in PAS-2 also were similar. CONCLUSION: Transfusion of PCs in PAS-2 significantly reduces the incidence of reactions. The 1-hour and 20-hour CCIs after transfusion of PCs in PAS-2 were significantly lower than the CCIs after transfusion of PCs in plasma. Because storage conditions of both PCs were found to be optimal, the decrease in CCIs after transfusion of PCs prepared in PAS-2 may be caused by rapid elimination of a subpopulation of P-selectin-positive platelets from the circulation.     

Co-localization of von Willebrand factor with platelet thrombi, tissue factor and platelets with fibrin, and consistent presence of inflammatory cells in coronary thrombi obtained by an aspiration device from patients with acute myocardial infarction

BACKGROUND: Detailed histochemical analysis of coronary thrombi obtained freshly from acute phase of myocardial infarction patients may provide information necessary to understand the mechanism of coronary occlusive thrombus formation. METHODS AND RESULTS: Coronary thrombi causing myocardial infarction were obtained from 10 consecutive patients of myocardial infarction in the acute phase, using a newly developed aspiration catheter. All the fixed specimens of coronary thrombi, by hematoxylin and eosin staining, were found to contain three major constituents, namely, platelets, densely packed fibrin and inflammatory cells, including polymorphonuclear and mononuclear cells, although their distribution in each specimen is totally heterogeneous. Immunohistochemical staining revealed the prominent presence of von Willebrand factor (VWF) at the sites of platelet accumulation, presence of tissue factor and platelets at the sites of deposition of fibrin fibrils. It also revealed the presence of CD16-, CD45- and CD34-positive cells, yet the functional roles of these cells have still to be elucidated. There are weak positive correlation between the number of inflammatory cells involved in the unit area of coronary thrombi specimen and the time of collection of the specimens after the onset of chest pain. CONCLUSIONS: In spite of various limitations, our results contain information suggesting the possible role of VWF in platelet-thrombus formation, possible important role played by tissue factor and activated platelets in the formation of fibrin fibrils, and the positive relationship between inflammatory cells migration and the formation of occlusive thrombi in human coronary arteries.     

WBC-reduced platelet concentrates from pooled buffy coats in additive solution: an evaluation of in vitro and in vivo measures

BACKGROUND: The use of a platelet additive solution (PAS-II, Baxter) may have benefits over plasma for storage of platelets. It was the aim of this study to develop a method to produce WBC-reduced platelet concentrates (PCs) in PAS-II with >240 x 10(9) platelets and <1 x 10(6) WBCs per unit, which can be stored for 5 days at pH >6.8 and that will give sufficient platelet increments after transfusion: a 1-hour CCI of >7.5 and a 20-hour CCI of >2.5. STUDY DESIGN AND METHODS: PCs were made from five pooled buffy coats and 250 g of PAS-II. After centrifugation the PCs were WBC-reduced with a filter (Autostop BC, Pall Biomedical) and stored in a 1000-mL polyolefin container. CCIs were assessed in stable hemato-oncologic patients after 5-day old PCs were transfused. RESULTS: Routinely produced PCs contained a median of 310 x 10(9) platelets (n = 5,363) with 3.5 percent containing <240 x 10(9) platelets, in a median volume of 320 mL (n = 11,834). The median number of WBCs was <0.03 x 10(6) (n = 694). The WBC count exceeded 1 x 10(6) in three PCs, but it was always <5 x 10(6), giving 99-percent confidence that more than 99.5 percent of the units will contain <1 x 10(6) WBCs. The pH remained >6.8 on Day 8, provided the concentration was below 1.1 x 10(9) platelets per mL (n = 32). After 28 transfusions in 28 patients, the 1-hour CCI was 12.6 +/- 4.3 (mean +/- SD, with 2/28 CCIs <7.5) and the 20-hour CCI was 8.9 +/- 5.6 (with 4/28 CCIs <2.5). Limitations of this study include the absence of a control group of patients receiving platelets stored in plasma and of in vivo radiolabeled survival studies, but a comparison of these data with previously published data suggested that the in vivo survival of platelets stored in PAS-II is less than that of platelets stored in plasma. CONCLUSION: The WBC-reduced PCs conformed to specifications. These WBC-reduced PCs could be stored at least 5 days with maintenance of pH, and they gave sufficient increments after transfusion to patients.     

Comparative in vitro evaluation of apheresis platelets stored with 100% plasma or 65% platelet additive solution III/35% plasma and including periods without agitation under simulated shipping conditions

BACKGROUND: A comparative study evaluated the retention of apheresis platelet (A‐PLT) in vitro properties prepared with PLT additive solution (PAS)‐III or 100% plasma and stored with continuous agitation (CA) and without continuous agitation (WCA). STUDY DESIGN AND METHODS: PLTs collected with the Amicus cell separator (Fenwal, Inc.) were utilized to prepare two matched components, each with approximately 4 × 10¹¹ PLTs. In the primary study, one component contained 65% PAS‐III/35% plasma and the other 100% plasma. Four storage scenarios were used, one with CA and three with periods without agitation under simulated shipping conditions. In vitro assays were used early and after 5 days of storage. RESULTS: pH levels after 5 days with CA were less with PAS‐III components than 100% plasma components, with levels always above 6.6 in any component. With CA, a number of other variables were reduced even early during storage with PAS‐III including morphology, extent of shape change, hypotonic stress response, adhesion, and aggregation. Storage WCA resulted in only a limited increase in the magnitude of the assay differences between PAS‐III and 100% plasma components. Periods WCA did not reduce the pH below 6.6. The thromboelastograph variable associated with the strengthening of clots by PLTs was essentially comparable with PAS‐III and plasma components throughout storage with CA or WCA. CONCLUSION: The data indicate that a 100% plasma medium provides for better retention of specific in vitro PLT properties, with CA and WCA, although the clinical significance of these in vitro decrements due to PAS‐III is unknown.