BACKGROUND

With buffy coat (BC) processing of whole blood (WB) donations, increase in WB storage time to facilitate overnight holding before the separation of blood components would be a logistically attractive development. This study undertakes a comparative in vitro characterization of blood components prepared from WB samples that were either processed within 8 hours or stored overnight at room temperature before processing by the BC method.

METHODS

The WB units (400 mL) collected were either processed within 8 hours (fresh blood) or stored overnight (overnight blood) at room temperature. WB units were separated into individual-component red blood cells (RBCs), BC, and plasma. The in vitro quality of these blood components (RBCs, pooled platelet concentrates [PCs], and plasma) was analyzed during storage.

RESULTS

Levels of 2,3-diphosphoglycerate (2,3-DPG) were found to be significantly lower immediately after processing, compared with the fresh WB samples, in RBCs that had been separated from an overnight-hold sample. However, this difference was not apparent after 14 days of storage. In pooled PCs, measurements for glucose, lactate, PO(2), PCO(2), extent of shape change, and hypotonic shock response were similar. The platelet yield in PCs prepared from an overnight-hold WB sample was significantly higher, while CD62P expression and annexin V binding were lower (p < 0.05). For frozen plasma (FP), no significant differences were observed for the coagulation factors (F)II, FVII, FV, F IX, FX, and FXI; fibrinogen; and von Willebrand factor content between the 8- and 24-hour FP. The FVIII was the component that was most sensitive to the prolongation of production time and it only had 80% of the activity of the 8-hour FP.

CONCLUSIONS

These data suggest that blood components (RBCs, pooled PCs, and FP) separated from WB that has been stored overnight at room temperature by the BC method are of acceptable quality.

BACKGROUND

We evaluated the effect of treating platelets (PLTs) using ultraviolet (UV)C light without the addition of any photosensitizing chemicals on PLT function in vitro and PLT recovery and survival in an autologous radiolabeled volunteer study.

METHODS

For in vitro studies, pooled or single buffy coat-derived PLT concentrates (PCs) were pooled and split to obtain identical PCs that were either treated with UVC or untreated (n = 6 each) and stored for 7 days. PLT recovery and survival were determined in a two-arm parallel autologous study in healthy volunteers performed according to BEST guidelines. UVC-treated or untreated PCs (n = 6 each) were stored for 5 days and were compared to fresh PLTs from the same donor.

RESULTS

There were no significant differences on Day 7 of storage between paired UVC-treated and control PC units for pH, adenosine triphosphate, lactate dehydrogenase, CD62P, CD63, PLT microparticles, and JC-1 binding, but annexin V binding, lactate accumulation, and expression of CD41/61 were significantly higher in treated units (p < 0.05). Compared with control units, the recovery and survival of UVC-treated PC were reduced after 5 days of storage (p < 0.05) and when expressed as a percentage of fresh values, survival was reduced by 20% (p = 0.005) and recovery by 17% (p = 0.088).

CONCLUSIONS

UVC-treated PLTs stored for 5 days showed marginal changes in PLT metabolism and activation in vitro and were associated with a degree of reduction in recovery and survival similar to other pathogen inactivation systems that are licensed and in use.

Flow cytometry can detect platelet activation (CD62p), aggregate formation, microparticle formation, and glycoprotein IIb/IIIa (GP IIb/IIIa) receptor occupancy in one sample at the level of single particles. We studied the effect of GP IIb/IIIa inhibitors on platelet activation with flow cytometry in vitro. Citrated whole blood was incubated with increasing concentrations of three different GP IIb/IIIa inhibitors (c7E3, DMP728, XJ757), then thrombin or adenosine diphosphate (ADP) was added, and after 1 minute the sample was fixed. Samples with thrombin but without c7E3 had a decrease in platelet count, from a mean of 260,000 platelets/microl to 56,000 platelets/microL, and aggregates increased. Samples with concentrations of c7E3 that resulted in 80% or more receptor blockade had no decrease in platelet count, and no aggregates were formed, but the number of CD62p-positive single platelets increased from 1200 to 7400 platelets/microL. The two other inhibitors (DMP 725, XJ757) or ADP instead of thrombin gave similar results. Microparticle formation did not change with platelet activation in the presence of a GP IIb/IIIa inhibitor. With small inhibitor doses resulting in <80% receptor blockade, the number of aggregates did not change or was even higher than that in samples without inhibitor. GP IIb/IIIa inhibitors do prevent aggregate formation but they do not prevent activation of platelets. With GP IIb/IIIa inhibition, more activated single platelets remain in the blood. One may expect an increasing number of circulating, activated platelets with the use of GP IIb/IIIa inhibitors.

With platelet activation, there is modulation of platelet surface molecule expression. In flow cytometric analyses of in vivo platelet activation, results are often confounded by activation induced in vitro by the preparative procedures. It is particularly important therefore to prevent or retard platelet activation as soon as possible after withdrawal of the blood sample. Taking blood into paraformaldehyde, or fixing the cells with paraformaldehyde as soon as possible after withdrawal, has been employed to prevent platelet activation in vitro, but paraformaldehyde-fixed platelets cannot be further used in functional studies. We investigated the efficacy of Diatube-H, a commercially available combination of platelet antagonists (theophylline, adenosine, and dipyridamole), in preventing or retarding platelet activation in vitro, along with its effects on modulation of platelet membrane glycoproteins (GP) and adhesion molecules. In contrast to blood taken into EDTA, blood taken into Diatube-H vacutainer tubes could be stored at room temperature for up to 4 hr prior to paraformaldehyde fixation without significant in vitro platelet activation, as measured by CD62P, CD63 and modulation of GPIb and GPIIbIIIa surface expression. Hence, paraformaldehyde fixation could be deferred for several hours, permitting transport of samples from distant sites. Studies of thrombin-induced platelet activation indicated that platelets taken into Diatube-H remained functional i.e. were able to be activated. Expression of the CD29, CD49b and CD31 adhesion molecules on the platelet surface was unaffected by storage in Diatube-H. The results suggest that Diatube-H may be a useful reagent for flow cytometric studies of platelets when the samples cannot be processed immediately.

The recently synthesized polyurea-nanoencapsulated surfactant-templated aerogels (X-aerogels) are porous materials with significantly improved mechanical strengths. Surface-wise they resemble polyurethane, a common biocompatible material, but their biocompatibility has never been investigated. As lightweight and strong materials, if X-aerogels also have acceptable biocompatibility, they may be used in many implantable devices. The goal of this study was to investigate their biocompatibility toward platelets, blood plasma, and vascular endothelial cells, in terms of cell activation and inflammatory responses. Platelets were incubated with X-aerogel and platelet activation was measured through CD62P and phosphatidylserine expression. Platelet aggregation was also measured. Contact with X-aerogel did not induce platelet activation or impair aggregation. To determine X-aerogel-induced inflammation, plasma anaphylatoxin C3a level was measured after incubation with X-aerogel. Results showed that X-aerogel induced no changes in plasma C3a levels. SEM and SDS-PAGE were used to examine cellular/protein deposition on X-aerogel samples after plasma incubation. No structural change or organic deposition was detected. Furthermore, X-aerogel samples did not induce any significant changes in vascular endothelial cell culture parameters after 5 days of incubation. These observations suggest that X-aerogels have a suitable biocompatibility toward platelets, plasma, and vascular endothelial cells, and they have potential for use in blood implantable devices.

OBJECTIVE

Hypothalamic-pituitary-adrenal system dysfunction, serotonergic system alterations, and enhanced platelet activity may contribute to the increased cardiac risk in depression. This exploratory study examined associations between cortisol parameters, platelet serotonin (5-HT) content, and platelet activity markers in patients with newly diagnosed major depression (MD) and/or Type 2 diabetes (T2DM) compared with healthy controls.

METHODS

We compared cortisol awakening response (CAR), diurnal decrease in salivary cortisol concentrations (slope), platelet 5-HT, and platelet markers (CD40, CD40 ligand [CD40L], soluble CD40L, CD62P, β-thromboglobulin, and platelet factor-4) in 22 T2DM patients, 20 MD patients, 18 T2DM patients with MD, and 24 healthy controls.

RESULTS

Platelet markers were elevated in MD (F(6,60) = 11.14, p < .001) and T2DM (F(6,60) = 13.07, p < .001). Subgroups did not differ in 5-HT or cortisol slope, whereas T2DM patients without depression had significantly lower CAR than did healthy controls (F(1,61) = 7.46, p = .008). In healthy controls, cortisol slope correlated with platelet activity for CD40 (r = -0.43, p = .048) and 5-HT was correlated with CD40L (r = 0.53, p = .007). In patients with both T2DM and MD, 5-HT and CD62P were correlated (r = 0.52, p = .033).

CONCLUSIONS

Increased platelet activity in T2DM and MD may play a role in the association between diabetes, depression, and coronary artery disease. The present data suggest that group differences in cortisol or 5-HT as well as group-specific associations of cortisol or 5-HT with platelet markers might be of limited importance in the shared pathways of T2DM and depression in the pathophysiology of coronary artery disease.

Activation of platelets leads to cytoskeletal assembly that is responsible for platelet motility and internal contraction. We have evaluated the involvement of the cytoskeleton in platelet activation by two strong agonists, collagen and thrombin. Activation was assessed by measuring changes in cytoskeletal assembly, externalization of activation-dependent markers and expression of procoagulant activity, and tyrosine phosphorylation of proteins, in both the absence and the presence of cytochalasin B. Activation of platelets with collagen and thrombin induced morphological changes and increased the expression of CD62P, CD63, glycoprotein IV, and binding of annexin V to platelets. Moreover, both activating agents induced actin polymerization, increased the association of other contractile proteins, and promoted tyrosine phosphorylation of multiple proteins, some of which were associated with the cytoskeleton. The presence of cytochalasin B blocked the previous events when collagen was used as the activating agent, although binding of annexin V still occurred. In contrast, platelet response to thrombin was not completely prevented by the presence of cytochalasin B. Thus, activation by collagen requires a functional cytoskeleton to trigger signaling through tyrosine phosphorylation and secretion. This is not the case for thrombin, which is capable of activating signaling mechanisms in the presence of strong inhibitors of cytoskeletal assembly. Moreover, the expression of a procoagulant surface in platelets still occurs even when platelet motility has been inhibited.

Several lines of evidence point to the potential role of nitric oxide (NO) in the pathophysiology, as well as in the therapy, of sickle cell disease (SCD). In this study, we compared the effects of NO on platelets from normal individuals and from patients with SCD. Three NO donors were used to deliver NO to platelets: sodium 2-(N, N-diethylamino)-diazenolate-2-oxide (DEANO), S-nitrosocysteine (CysNO) and sodium trioxdintrate (OXINO or Angeli's salt). ADP-induced platelet aggregation, CD62P expression, PAC-1 binding and calcium elevation were evaluated in paired studies of normal and SCD subjects. DEANO significantly reduced aggregation in SCD platelets compared with normal platelets. DEANO similarly reduced the extent of CD62P expression in SCD platelets. All NO donors reduced PAC-1 binding, but there were no significant differences between platelets from normal or SCD subjects. Calcium elevation, as induced by ADP, was not altered by the presence of NO donors. However, when platelets were stimulated with thrombin, there was an increased initial response of SCD platelets compared with normal platelets. Taken together, these data suggest that the mode of NO delivery to platelets may produce various physiological responses and the optimization of NO delivery may contribute to reducing platelet aggregation in sickle cell disease.

BACKGROUND

Whole blood (WB) must be refrigerated within 8 hours to optimize Factor VIII: C yield, but chilled platelets (PLTs) are rapidly removed from circulation and cannot be used clinically. It is logistically preferable to hold WB overnight at room temperature for next-day processing. We compared in vitro quality of PLT-rich plasma (PRP)-derived PLT concentrates (PCs) from fresh versus overnight-held WB.

METHODS

Four units of WB were pooled and split to prevent donor-dependent differences. One unit was processed immediately; three others were held at room temperature and processed after 24 to 26 hours. After soft-spin centrifugation, PRP was separated from the red blood cells. PRP was hard-spun to make PLT-poor plasma, and the PLTs were resuspended in approximately 60 mL of plasma and stored for 7 days (n = 8 paired experiments by two blood centers).

RESULTS

After overnight hold, the PLT concentration was 1.37 × 10(9) ± 0.19 × 10(9) /mL versus 1.03 × 10(9) ± 0.32 × 10(9) /mL for freshly prepared PCs (p < 0.05). pH and glucose were significantly lower and lactate higher for overnight-held units on Day 1, but by Day 5, the differences had disappeared. Hypotonic shock response was initially better, 73 ± 14% for overnight-held versus 53 ± 12% for freshly processed (p < 0.001), but this difference also disappeared during storage. Activation marker CD62P was not different.

CONCLUSIONS

In vitro storage conditions produce immediate differences after preparation, which disappear throughout storage. PCs from overnight-held WB have similar in vitro variables as from freshly processed WB. These findings warrant confirmation in clinical trials, but underscore the possibility of use of these PCs as being equivalent to those obtained from freshly processed WB.

OBJECTIVE

Mirasol pathogen reduction technology (PRT) for platelet concentrates uses riboflavin and ultraviolet light. Previously, we described increased metabolism and activation for PRT platelets stored in 100% plasma. To improve platelet quality, we resuspended platelets in a mixture of plasma and platelet additive solution (PAS).

METHODS

Single-donor platelets were resuspended in plasma and split into an untreated control and a PRT-treated single product. One hundred and fifty millilitre PAS (SSP+) was added to both. Over 7 days, we assayed pH, glucose consumption-, lactate production rate and CD62p with and without TRAP.

RESULTS

On day 5, PRT units showed a significantly lower pH (7.087 +/- 0.105 vs. 7.288 +/- 0.200) accompanied by a higher lactate production (0.104 +/- 0014 vs. 0.063 +/- 0.017 mmol/10(12)/h) and glucose consumption rate (0.039 +/- 0005 vs. 0.028 +/- 0.009 mmol/10(12) platelets/h). CD62p expression was higher in treated units (44.5 +/- 13.0 vs. 16.5 +/- 7.6%).

CONCLUSIONS

In comparison to PRT platelets resuspended in 100% plasma, a mixture of plasma and PAS improves pH and platelet metabolism but not platelet activation. Prolonged shelf-life for up to 7 days may be possible

BACKGROUND

Filtered PLT concentrates (PCs) were prepared in plasma pooling three (for children) or six buffy coats (BCs; for adults) after holding them a maximum of 4 hours (blood bags collected in the afternoon) or 18 hours (blood bags collected in the morning).

METHODS

With flow cytometry, PCs prepared after holding BCs 4 or 18 hours were compared. BCs removed from whole-blood donations in quadruple bag packs ("top-top") were held 4 or 18 hours before pooling them with a sterile connecting device. After the BCs were centrifuged, the supernatant was transferred through a BC filter (Autostop, Pall Medical) to a CLX bag. Samples for analysis were collected from the whole-blood bag, BCs, and PCs immediately after preparation and after 1, 3, 5, and 7 days of storage on a flat-bed agitator at 22 +/- 2 degrees C. The main PLT membrane glycoproteins (GPs, IIb-IIIa, IV, and Ibalpha), some of their ligands (fibrinogen, fibronectin, and VWF), activation-dependent antigens (CD62P and CD63), and procoagulant activity markers (annexin V and bound coagulation FV-Va) have been studied.

RESULTS

In the 12 PCs (six pools of 3 units each group) studied, a minor increase in activation markers during preparation was observed. During the storage, a significant increase in the expression of GPIIb-IIIa, CD62P, CD63, annexin V, and FVa was measured. After 5 days of storage, only the percentage of PLTs with bound fibrinogen was significantly greater in PCs prepared after holding BCs for 4 hours.

CONCLUSIONS

In PCs prepared after holding BCs 4 or 18 hours before pooling and filtering, only a minor significant difference in the percentage of PLTs with bound fibrinogen was found after 5 days of storage. This difference is probably of little, if any, transfusional significance.

The effect of an arterial filter on visceral emboli was quantified with autologous indium-111 labeled platelets (INPLT) during cardiopulmonary bypass (CPB) in Yorkshire pigs. Biodistribution of INPLT was determined in 12 control pigs (30-35 kg, unoperated control [n = 6] and sham operated control [n = 6]). CPB was carried out with (n = 6) and without (n = 6) an arterial filter in 12 pigs at a flow rate of 2.5-3.5 L/min. Platelets labeled with In-111 tropolone (650-780 microCi) were injected intravenously 24 hr before CPB. All pigs were systemically heparinized (activated coagulation time>> 400 sec); CPB was instituted with a roller pump, an extraluminal blood flow oxygenator (Bentley Univox, 1.8 m2), and an arterial filter (0.25 m2) and continued for 3 hr. Platelet kinetics, pooling, and counts were monitored by a Geiger probe and a Coulter counter. The thrombi in the oxygenator and arterial filter and emboli in viscera and brain were imaged with a gamma camera and measured with an ion chamber and gamma counter. Percentage of INPLT (mean +/- SD) in organs, tissues, and components of the circuit in four groups of pigs was calculated. Flow cytometry with antibodies to CD61 (GPIIIa) and CD62P (GMP-140: control) of porcine platelets was carried out with blood samples taken before, during, and after CPB for estimation of circulating platelet aggregates and platelet microparticles. Pulmonary, renal, cardiac, and cerebral emboli in pigs undergoing CPB with and without a filter were similar (p < 0.1). The amount of filter adherent thrombi was small (0.04 +/- 0.01%); oxygenator adherent thrombus in both groups was similar (p < 0.1). Emboli were found in the cerebral medulla, hippocampus, and posterior cerebral cortex in both groups. During CPB, the arterial filter functioned minimally as a trap for platelet thrombi detached from the oxygenator and circulating emboli. Flow cytometry of blood demonstrated the shift of equilibria from single platelets to platelet aggregates and microparticles during CPB and their gradual reversal to single platelets after CPB; the loosely adherent emboli disaggregated and further shifted these equilibria to single platelets and smaller aggregates, probably through the action of endogenous nitric oxide and prostacyclin. The emboli were trapped in organs and tissues and microparticles were sequestered by the reticuloendothelial system.

OBJECTIVE

To investigate effects of ethanol on activity markers of atherosclerosis in an in vitro endothelial cell model.

METHODS

After 24 h incubation with ethanol (0.0095%), human umbilical vein endothelial cells were stimulated for 1 h with lipopolysaccharide, and were then incubated in direct contact with activated platelets. Following this incubation, the expression of CD40L and CD62P on platelets, and the expression of intercellular adhesion molecule-1 (ICAM-1), vascular cell adhesion molecule-1 (VCAM-1), urokinase plasminogen activator receptor (uPAR), and membrane-type 1 matrix metalloproteinase (MT1-MMP) on endothelial cells were measured by flow cytometry.

RESULTS

The increased expression of VCAM-1 and uPAR on endothelial cells by proinflammatory stimulation with activated platelets was significantly reduced through pre-incubation with ethanol (P < 0.05). Furthermore, platelets in direct contact with ethanol and with endothelial cells pre-incubated in ethanol showed a significant reduction in their CD40L expression (P < 0.05). Ethanol had no significant effect on ICAM-1 and MT1-MMP expression on endothelial cells.

CONCLUSIONS

Ethanol directly attenuates platelet activation and has significant endothelial cell-mediated effects on selected markers of atherosclerosis in vitro. These findings underline possible protective effects of ethanol on atherosclerosis.

Plasma concentrations of the circulating adhesion molecules ICAM-1 (CD54), VCAM-1 (CD106) were determined in 31 women with pre-eclampsia, 9 women with HELLP syndrome, and 13 women with transient pregnancy induced hypertension (PIH). Data were compared with a control group of 157 healthy pregnant women of the same gestational age. Furthermore, concentrations of circulating E-selectin (CD62E), P-selectin (CD62P), and PECAM-1 (CD31) were determined in a subpopulation of 17 women with pre-eclampsia. Plasma concentrations of circulating ICAM-1, VCAM-1, E-selectin, and PECAM-1 were significantly elevated in women with pre-eclampsia compared to healthy control pregnant women. Circulating ICAM-1 and VCAM-1 levels were also significantly elevated in the pre-eclampsia group compared to women with PIH. Concentrations of circulating P-selectin varied strongly in all experimental groups (SD>> 70% of the mean), most likely reflecting various degrees of thrombocyte degranulation in the individual samples. Finally, longitudinal profiles of cICAM-1 and cVCAM-1 concentrations were determined in 123 healthy pregnant women between the 16th and the 42nd week of gestation. This analysis identified cICAM-1 and cVCAM-1 as tightly regulated plasma parameters that varied in a small concentration range. Concentrations of cICAM-1 and cVCAM-1 did not vary during pregnancy and the determined concentrations corresponded to the reported reference levels of nonpregnant individuals.

BACKGROUND

Platelet-derived extracellular vesicles (PL-EVs) are present in plateletpheresis concentrates (PCs) and may influence the quality of PCs. The aim of the study was to analyze PC-derived PL-EVs and to correlate them with standard quality control (QC) variables of PCs and with donor-specific laboratory variables.

METHODS

PL-EVs were analyzed by standard as well as advanced high-sensitivity flow cytometry (FCM) and nanoparticle tracking analysis. A hematology analyzer was applied to the determination of platelet (PLT) count and immature PLT fraction (IPF). Functional capacity of PLTs (CD62P in response to thrombin receptor-activating peptide 6 activation) was measured by FCM. All in vitro measurements were carried out on Day 0 and on Day 5. Altogether, a total of 42 PC samples, 15 irradiated on Day 0, were investigated.

RESULTS

Externalization of CD62P, as an indicator of intact PLT function, significantly decreased during in vitro PLT senescence and CD62P expression inversely correlated with increased PL-EV levels. Interestingly, in fresh PCs a significant correlation was found between PL-EVs and different hemapheresis instruments, duration of apheresis, and IPF count in peripheral blood of the donor before apheresis. In senescent PCs, the body mass index of donors inversely correlated with the PL-EV counts.

CONCLUSIONS

Loss of PLT function in PCs was associated with increased PL-EV levels. Shedding of PL-EVs depends on shear stress influenced by different hemapheresis settings and diverse preanalytical conditions of donors. PL-EV analysis may stimulate new quality and apheresis strategies for more vital PLTs for transfusion.

The human platelet antigen-1 (HPA-1, Pl(A)) polymorphism has been proposed to influence the inhibitory actions of abciximab. Thus, we hypothesized that this polymorphism might also be the cause for paradoxical activation of platelets by GPIIb/IIIa inhibitors. The effects of abciximab (1-10 microg/ml), tirofiban (3-30 nM), or eptifibatide (0.3-3 microg/ml) on basal and ADP (3 microM)-induced CD62P externalization were measured in n=62 healthy blood donors and n=177 patients with stable coronary artery disease. All subjects were genotyped for the human platelet antigen-1 (HPA-1, Pl(A)) polymorphism by GALIOS(R) and fluorescence correlation spectroscopy. Although a significant platelet hyperreactivity was observed in the patients, the HPA-1 genotype did not influence basal or ADP-induced CD62P expression. A moderate (twofold) stimulation of CD62P expression by abciximab but not by tirofiban or eptifibatide was observed in one patient. Interestingly, this patient carried the HPA-1 b/b genotype. In no other subject any activation of platelets by GP IIb/IIIa inhibitors was observed and there were no statistically significant differences between HPA-1 genotypes with respect to the effects of GP IIb/IIIa inhibitors on basal or ADP-stimulated CD62P expression. It is concluded that paradoxical platelet activation by abciximab is a rare (<2%) phenomenon. HPA-1 b/b genotype might be a contributing factor but clearly does not predict platelet activation by GP IIb/IIIa inhibitors.

Recently, it has been reported that ankylosing spondylitis (AS) was characterised by endothelial dysfunction and the development of atherosclerotic complications. In this study, we evaluated platelet and endothelial activation parameters in AS patients. Fiftynine AS patients and 22 healthy controls were included. The clinical features and acute phase parameters were evaluated. In all patients and healthy controls, platelet-monocyte complexes (PMC), platelet-neutrophil complexes, basal and ADP-stimulated P-selectin (CD62P) expression were determined by flow cytometry; soluble E-selectin (sE-selectin) and soluble CD40L (sCD40L) were determined by ELISA. AS patients were divided into two groups as active and inactive by using BASDAI. In 15 AS patients, the evaluated parameters were assessed before and after 12 weeks of anti-TNF therapy. PMC and sCD40L levels in AS patients were significantly higher than in the control group (P values 0.013 and 0.016). The evaluated variables were similar in active and inactive AS groups (P>> 0.05). There were no significant changes in platelet and endothelial activation parameters in AS patients after anti-TNF therapy (P>> 0.05). Platelet activation which is reflected by high levels of PMC and sCD40L might be responsible for the increased frequency of atherosclerosis in AS. The platelet activation in our AS patients was not associated with disease activity and did not improve after anti-TNF therapy.

This study was undertaken to evaluate the effects of thin film hyaluronic acid and dextran surface coatings to blunt cellular activation in a laboratory model of extracorporeal blood circulation. The inner lumen surface of polyurethane (PU) and poly(vinyl) chloride (PVC) tubing was grafted with hyaluronic acid and dextran. Surfaces were characterized for the presence of the grafted layer using ellipsometry, atomic force microscopy (AFM), and X-ray photoelectron spectroscopy (XPS). Persistence of the surface layer was maintained for up to 5 days of continuous exposure to shear flow using a Chandler loop apparatus. The Chandler loop method was used to study human whole blood activation activity. Whole blood aggregometry and flow cytometry measures of CD18, CD62L, CD62P, Annexin V and myeloperoxidase performed on blood samples exposed to the tubing for up to three hours were complemented by scanning electron microscopy (SEM) analysis of adherent cells and state of activation. In these studies commercial hospital products and uncoated PVC and PU tubes were used as controls. We found that hyaluranized PU and PVC conferred the greatest resistance to blood activation and that dextranization of the PU and PU tubing also provided significant diminution of the bioresponses measured. Based on our findings, we suggest that surface coating with hyaluronic acid or dextran acts as a potent shield from blood cellular activation during forms of extracorporeal circulation.

BACKGROUND

The aim of this study was to demonstrate how ultrastructural morphology of platelets stored in different media correlate with the appearance of particular activation markers on their cell surface.

METHODS

Concentrates of buffy coat-derived platelets were stored in plasma or a glucose-free citrate-acetate-NaCl platelet additive solution (PAS2, Baxter Healthcare Corp.). Activation markers on platelets were measured by flow cytometry and compared with changes in the platelet cell surface as demonstrated by electron microscopy. Levels of the vasoactive cytokines vascular endothelial growth factor (VEGF) and RANTES (regulated upon activation, normal T-cell expressed and secreted) were determined in the storage medium of the platelet concentrate.

RESULTS

The activation markers CD62P and CD63 and the binding of thrombospondin measured by flow cytometry were expressed to a higher extent in the PAS2 group compared with the plasma group. The difference reached significance on Day 3 (CD62P: 66.37 +/- 2.44 vs. 37.83 +/- 2.03, p < 0.001; CD63: 42.11 +/- 3.29 vs. 34.84 +/- 2.04, p < 0.05; and thrombospondin binding: 18.84 +/- 3.9 vs. 13.98 +/- 3.87, p < 0.001, respectively). The form factor that is related to changes of the platelet shape was determined by image analysis and correlated significantly with the cell surface expression of CD62P (p < 0.001) and with CD63 (p < 0.05) and with thrombospondin binding (p < 0.05). The chemokines VEGF and RANTES were measured at higher levels in the PAS2 group.

CONCLUSIONS

With exception of baseline activation probably due to necessary handling procedures, platelets remain relatively unaltered and more stable in plasma in comparison to storage in PAS2.

BACKGROUND

A novel short-wave ultraviolet light (UVC) pathogen reduction technology (THERAFLEX UV-Platelets; MacoPharma, Mouvaux, France) without the need of any additional photoactive reagent has recently been evaluated for various bacteria and virus infectivity assays. The use of UVC alone has on the one hand been shown to reduce pathogens but may, on the other hand, have some impact on the platelet (PLT) quality. The purpose of this study was to determine the potential effects on PLT quality of pathogen inactivation treatment using the novel UVC method for PLT concentrates.

METHODS

Buffy-coat-derived PLTs suspended in SSP+ were irradiated with UVC light in plastic bags (MacoPharma) made of ethyl vinyl acetate, considered to be highly permeable to UVC light. The UVC-treated (test, n=8) as well as the untreated (reference, n=8) PLT units were stored in PLT storage bags composed of n-butyryl, tri n-hexyl citrate-plasticized polyvinyl chloride (MacoPharma) on a flat bed agitator for in vitro testing during 7 days of storage.

RESULTS

No significant difference in PLT counts and lactate dehydrogenase between the groups was detected. During storage, glucose decreased more and lactate increased more in the test units. Statistically significant differences were found for glucose (P<0·01) and lactate (P<0·05) on day 7. ATP levels were higher (P<0·01 from day 5) in the reference units. With exception of day 7 (P<0·01 reference vs. test), hypotonic shock response reactivity was not different between groups. Extent of shape change was lower (P<0·01), and CD62P (P<0·05 day 5) was higher in the test units. CD42b and CD41/61 showed similar trends throughout storage, without any significant difference between the units. pH was maintained at >6·8 (day 7) and swirling remained at the highest level (score = 2) for all units throughout storage.

CONCLUSIONS

Our results suggest that irradiation with UVC light has a slight impact on PLT in vitro quality and appears to be insignificant with regard to current in vitro standards.

BACKGROUND

Patients who have experienced anaphylactic transfusion reactions receive washed platelet (PLT) concentrates (PCs) where the plasma has been substituted with a PLT additive solution. This study compares the in vitro quality of PCs washed at the beginning of the storage period (Day 1) to PCs washed at the end of storage (Day 7).

METHODS

PLTs were prepared by the buffy coat procedure. Two concentrates were pooled and then split to obtain an identical pair of PCs. One of the PCs was washed with T-Sol on Day 1 and the other on Day 7 of storage. Swirling, blood gases, and metabolic variables were analyzed before washing. Analyses of surface expression of CD62P and coagulation by free oscillation rheometry (FOR) were performed before and after washing.

RESULTS

pH was acceptable in all PCs. Washing on Days 1 and 7 increased the CD62P surface expression. The FOR variables clotting time and clot retraction were not influenced by washing on either day. Washing resulted in a decrease in the number of PLTs and the decrease was larger on Day 7 compared to Day 1.

CONCLUSIONS

PLTs washed on Days 1 and 7 of storage are effected by washing in a similar manner. However, a larger loss of PLTs occurred during washing on Day 7.

BACKGROUND

Turbulent blood flow caused by arterial stenosis can induce platelet activation and aggregation, which subsequently participate in arterial thromboembolic events.

OBJECTIVE

To test the hypothesis that platelet activation (expressed by CD62p) is enhanced in cerebral vs systemic circulation in patients with severe internal carotid artery (ICA) stenosis.

METHODS

Platelet CD62p expression was prospectively measured using flow cytometry in 35 consecutive symptomatic patients with severe ICA stenosis and in 20 at-risk control subjects who underwent both coronary and cerebral angiographic studies due to angina pectoris and suspicious vertebral artery or intracranial artery stenosis. The CD62p expression was also evaluated in 20 healthy subjects. Blood samples were first drawn from the right internal jugular vein (cerebral circulation) and right femoral vein (systemic circulation) before extra- and intracerebral angiographic examination of both patients and at-risk control subjects and again at 40 minutes after ICA stenting. Clopidogrel was administered to the patients following the second blood sampling.

RESULTS

Systemic CD62p expression was higher in patients than in both the healthy and at-risk control subjects (both p < 0.0001). Additionally, cerebral CD62p expression was higher in patients than in at-risk control subjects (p < 0.0001) prior to intervention. Moreover, CD62p expression was higher in cerebral circulation than in systemic circulation in the patients (p < 0.0001) before ICA stenting. However, CD62p expression was less enhanced in cerebral circulation than in systemic circulation after ICA stenting (p < 0.0001). Furthermore, CD62p expression was decreased after 3 months of follow-up (p < 0.0001).

CONCLUSIONS

Compared to systemic CD62p expression, cerebral CD62p expression was more enhanced prior to ICA stenting and was less enhanced after ICA stenting in patients with severe ICA stenosis.

BACKGROUND

Cancer and stroke, which are known to be associated with one another, are the most common causes of death in the elderly. However, the pathomechanisms that lead to stroke in cancer patients are not well known. Circulating extracellular vesicles (EVs) play a role in cancer-associated thrombosis and tumor progression. Therefore, we hypothesized that cancer cell-derived EVs cause cancer-related coagulopathy resulting in ischemic stroke.

METHODS

Serum levels of D-dimer and EVs expressing markers for cancer cells (epithelial cell adhesion molecule [CD326]), tissue factor (TF [CD142]), endothelial cells (CD31+CD42b-), and platelets (CD62P) were measured using flow cytometry in (a) 155 patients with ischemic stroke and active cancer (116 - cancer-related, 39 - conventional stroke mechanisms), (b) 25 patients with ischemic stroke without cancer, (c) 32 cancer patients without stroke, and (d) 101 healthy subjects.

RESULTS

The levels of cancer cell-derived EVs correlated with the levels of D-dimer and TF+ EVs. The levels of cancer cell-derived EVs (CD326+ and CD326+CD142+) were higher in cancer-related stroke than in other groups (P<0.05 in all the cases). Path analysis showed that cancer cell-derived EVs are related to stroke via coagulopathy as measured by D-dimer levels. Poor correlation was observed between TF+ EV and D-dimer, and path analysis demonstrated that cancer cell-derived EVs may cause cancer-related coagulopathy independent of the levels of TF+ EVs.

CONCLUSIONS

Our findings suggest that cancer cell-derived EVs mediate coagulopathy resulting in ischemic stroke via TF-independent mechanisms.

In extracorporeal circulation, blood is affected by artificial biomaterials and shear forces. We investigated the effects of peripheral blood progenitor cell (PBPC) apheresis on the kinetics and level of platelet membrane antigen expression in 11 breast cancer and 13 testicular cancer patients. After mobilization with rhG-CSF, continuous-flow apheresis was performed. Expression of structural antigens CD41a and CD42b and activation-dependent antigens CD62p, CD63, and fibrinogen was analyzed by flow cytometry at fixed time intervals. Initial changes occurred in all of the antigens within minutes, followed by a progressive increase in the mean channel fluorescence intensities (MCFI) of CD62p from 26 +/- 8 (mean +/- SD) to 73 +/- 29 (p < 0.05), CD63 from 22 +/- 5 to 51 +/- 16 (p < 0.05) and antifibrinogen from 120 +/- 20 to 356 +/- 154 (p < 0.05). In contrast, CD41a and CD42b fluorescence decreased during apheresis (p < 0.05 for both). The more rapid sequestration of P-selectin-expressing platelets known to occur during extracorporeal PBPC apheresis suggests that platelet activation may be associated with the loss of platelets during this procedure. In addition, alteration of platelet surface antigens increases thrombogenic potential and may reduce the in vivo efficacy of the platelet hemostatic potential.