In vitro and in vivo effects of potassium and magnesium on storage up to 7 days of apheresis platelet concentrates in platelet additive solution

BACKGROUND AND OBJECTIVE: Prolonged storage of platelets up to 7 days provides improved availability, logistical management and decreased wastage. Beside methods of bacterial detection, addition of magnesium and potassium to the platelet storage solution (SSP+) may further improve the quality of platelets with extended storage. MATERIALS AND METHODS: Apheresis platelets from 10 donors were divided and stored in two different platelet additive solutions (PAS) (Intersol and SSP+) for a paired comparison. A variety of in vitro platelet function and metabolic assays were performed both on day 1 and after 7 days of storage. For in vivo study, platelets were labelled with either (111)Indium or (51)Chromium after 7 days of storage and were injected into the corresponding donor. Serial blood samples were drawn for recovery and survival measurements. RESULTS: In vitro parameters for SSP+ showed significantly reduced glycolysis (lower glucose consumption and decreased production of lactate), a higher hypotonic shock response (HSR) and the extent of shape change reactivity and a lower degree of platelet activation by means of RANTES (regulated on activation, normal, T cell-expressed, and secreted), CD62p and CD63 expression. Platelet recovery on day 7 was higher for Intersol as compared to SSP+, 65 +/- 11 vs. 53 +/- 13% (P = 0.023), and survival showed no difference 4.2 +/- 1.9 vs. 3.6 +/- 1.4 days. CONCLUSION: In vitro characteristics of platelets stored in PAS with addition of potassium and magnesium indicated higher quality, but this could not be verified by the in vivo parameters by means of recovery and survival.     

In vivo and in vitro effects of different anaesthetics on platelet function

Different effects of thiopental, propofol and sevoflurane on platelets have been reported. Patients undergoing thyroid surgery were anaesthetized with thiopental-fentanyl-sevoflurane (n = 11) or propofol-fentanyl-sevoflurane (n = 9). Platelet aggregation and thromboxane A2 generation were studied at baseline, and at the end of anaesthesia induction and surgery. Dose-response experiments were also performed in vitro with single agents. Thiopental-fentanyl-sevoflurane significantly reduced collagen-induced aggregation by the end of induction, while ADP-induced aggregation and thromboxane generation were unaffected. Propofol-fentanyl-sevoflurane had no effect on platelets. Thiopental dose-dependently inhibited platelets in vitro, while fentanyl or propofol did not. In conclusion, thiopental reduces platelet function both ex vivo and in vitro and propofol might be considered haemostatically safer.     

Survival in vivo of platelets stored for 48 hours in the buffycoat at 4°C compared to platelet rich plasma stored at 22°C

Summary High speed centrifugation allows separation of whole blood into cell free plasma, a buffy coat and leukocyte poor red cells. The buffy coat can be used for the preparation of platelet concentrates. High lactate production at 22°C requires storage of the buffy coat at 4°C. Survival in vivo of platelet concentrates prepared from buffy coats stored at 4°C for 48 h (BC-PC) was compared with the survival in vivo of platelet concentrates from platelet rich plasma stored at 22°C for 48 h (PRP-PC). Both methods were studied in the same healthy volunteers (n=8) using⁵¹Cr labeled autologous platelets. The mean ±SD recovery 15 min after reinfusion of the BC-PC was 30.5%±13.3% and for PRP-PC 41.4%±7.9% (p<0.0001). The survival in vivo for BC-PC was 2.4 days ±0.4 days and for PRP-PC 7.0 days ±1.4 days (p<0.0001).Since the survival in vivo is significantly less for platelets derived from the buffy coat stored at 4°C, we advocate storage of platelets at 22°C.     

Nonshivering thermogenesis in adult and aged C57BL/6J mice housed at 22°C and at 29°C

Twelve- and 28-month-old C57BL/6J male mice were housed either at room temperature of 22°C or at thermoneutrality (29°C) during the two months prior to experiments. Acute experiments were conducted under anesthesia, myorelaxation, and artificial ventilation. We recorded efferent electrical impulse activity in one of the sympathetic nerves innervating the interscapular brown adipose tissue in response to acute cold stimulation, when body temperature was lowered 7.5°C below control level. In separate experiments we measured O₂ consumption and CO₂ production and calculated the nonshivering thermogenesis. We also measured the concentration of uncoupling protein in interscapular brown adipose tissue before and after three-hour cold stress. In aged mice, both sympathetic nervous activity and nonshivering thermogenesis were lower in animals housed at thermoneutrality (29°C) than in mice housed at 22°. Among mice maintained at 22°C, but not at thermoneutrality, aged animals had greater nonshivering thermogenesis and greater cold induced concentration of uncoupling protein in the brown adipose tissue than adults. Sympathetic nervous outflow to brown adipose tissue was always greater in aged mice, regardless of the temperature of acclimation. We concluded that aged mice, housed at 22°C, showed the changes in nonshivering thermogenesis associated with cold acclimation. However, an increased sympathetic outflow to brown adipose tissue in aged animals reflects an age-related elevation of the tone and responsiveness of the sympathetic nervous system.     

Dynamic platelet function is markedly different in patients with cancer compared to healthy donors

Despite a fivefold increased risk of thromboembolism in patients with cancer, the mechanism of arterial thromboembolism is poorly understood. To address this, we investigated platelet function in cancer patients and healthy controls using an assay that mimics the arterial vasculature. Blood samples from cancer patients (n = 36) and healthy controls (n = 22) were perfused through custom-made parallel-plate flow chambers coated with von Willebrand factor (VWF) under arterial shear (1,500 s^?1). Multiparameter measurements of platelet interactions with the immobilized VWF surface were recorded by digital-image microscopy and analyzed using custom-designed platelet-tracking software. Six measured parameters that characterize in detail the surface motion and surface binding of several hundred platelets per blood sample differed significantly in those with cancer from the healthy donors. In particular, it was found that patients with cancer had decreased numbers of platelets interacting, translocating and adhering to VWF. There were also reductions in the speed and distances that platelets traveled on VWF in comparison to healthy controls. Platelet function differed between those with early-stage cancer compared to those with later stage cancer. Patients with advanced cancer had an increased number of platelets stably adhering to VWF and greater platelet surface coverage after a given time of interaction. To the best of our knowledge, our results demonstrate for the first time that dynamic platelet function is markedly different in patients with cancer compared to healthy donors.     

Platelet storage solution improves the in vitro function of preserved platelet concentrate

BACKGROUND AND OBJECTIVES: Stored platelets develop biochemical lesions, manifest as depressed haemostatic function, clot retraction and wound healing. ViaCyte trade mark, a proprietary experimental preservative solution (comprising D-ribose, D-glucose, Hanks solution, Hepes solution, bovine serum albumin, tic anticoagulant peptide and sterile water), was tested in comparison with the presently accepted storage solution, citrate-dextrose-phosphate-plasma (CDP-P), to evaluate its ability to preserve platelet function during storage. MATERIALS AND METHODS: Platelets stored in ViaCyte and platelets suspended in CDP-P were transferred to polypropylene tubes with PL732 covers and analysed for adenine nucleotide levels (ATP molecules), in vitro agonist-mediated P-selectin expression and aggregation. RESULTS: After 5 days of storage at room temperature, 12.2% of platelets stored in ViaCyte exhibited P-selectin expression at rest, and 64.2% exhibited P-selectin expression upon activation with thrombin challenge, an increase of 52%. Platelets stored in CDP-P exhibited 44.4% P-selectin expression at rest, suggesting significant activation during storage, and thrombin stimulation resulted in P-selectin expression of 47.9%, an increase of only 2.5% (P< or =0.002, untreated vs. treated). ViaCyte also maintained ATP levels throughout the storage period, while these levels became depressed in platelets stored in CDP-P (P< or =0.02, untreated vs. treated). Storing platelets in the experimental preservative solution maintained their ability to aggregate, while control platelets lost their ability to aggregate in response to agonist. CONCLUSIONS: ViaCyte appears to protect platelets during storage, reflected by a low level of induced lesions. Platelets stored in ViaCyte maintain energy levels at their resting state, which preserves their ability to aggregate and secrete granule contents, and ensures the availability of additional platelets for activation upon in vitro challenge.     

High-yield platelet units revealed immediate pH decline and delayed mitochondrial dysfunction during storage in 100% plasma as compared with storage in SSP+

Background and Objectives Initial elevated and prolonged high carbon dioxide levels related to mitochondrial dysfunction are recently being suggested as a contributing factor to rapid pH decline in platelet (PLT) units. The use of different storage environments may influence this phenomenon. This study has two objectives (i) to investigate the relationship of mitochondrial function and apoptotic events with different storage environments capability of pH control and (ii) to examine the cause and relationship between pH decline in PLT units, carbon dioxide levels and mitochondrial function. Materials and Methods Platelet units were prepared for storage in (A) 70% SSP+, 300–400 × 10⁹/unit, (B) 70% SSP+, 550–600 × 10⁹/unit, (C) 100% plasma, 550–600 × 10⁹/unit, and (D) additional 100% plasma, >600 × 10⁹/unit. In vitro variables including mitochondrial function (JC‐1), reactive oxygen species (ROS) and caspase 3 activity were analysed on days 2, 5 and 7. Results Glucose/lactate was higher, pH, ATP, Hypotonic shock response (HSR) and extent of shape change (ESC) decreased (P < 0·001 on day 7), CD62P (P < 0·001 on day 7) increased, the JC‐1‐positive PLTs were lower (P < 0·001 on day 7), and ROS was higher (P < 0·001 days 2–7) in the plasma (C) units as compared with the SSP+ (A) and (B) units. All plasma (D) units showed rapid pH and pCO₂ decline from day 2 but by means of >80% maintenance of mitochondrial function until day 7. Conclusions The use of SSP+ instead of plasma may reduce the risk of triggering pro‐apoptotic events in high‐yield PLT units. A rapid decline in pH in PLT units cannot be explained with initial elevated and prolonged high carbon dioxide levels and mitochondrial dysfunction.     

A randomized study of buffy coat platelets in platelet additive solution stored 1–5 versus 6–7 days prior to prophylactic transfusion of allogeneic haematopoietic progenitor cell transplant recipients

Background and Objective  Storage of platelets > 5 days provides improved availability, logistical management and decreased outdating. Promising results on in vitro parameters and on in vivo post-transfusion recovery and survival of autologous platelets in healthy volunteers have earlier been shown. To provide additional verification, randomized patient transfusion studies are needed.
Materials and Methods  Sixty allogeneic haematopoietic progenitor cell transplant recipients were randomized to receive buffy-coat (BC) platelets stored in platelet additive solution (PAS) for 1–5 days the first time a prophylactic transfusion was needed after transplantation, followed the second time by platelets stored for 6–7 days or vice versa. The corrected count increment (CCI) for 1 and 24 h were calculated.
Results  CCI 1 h and CCI 24 h were higher for platelets stored 1–5 days as compared to 6–7 days, 10·4 ± 5·1 vs. 7·4 ± 3·8 ( P <  0·001) and 5·4 ± 4·1 vs. 2·6 ± 2·6 ( P <  0·001), respectively. Time to next platelet transfusion was significantly longer after a transfusion of platelets stored for 1–5 days as compared to platelets stored for 6–7 days: 2·2 ± 1·1 vs. 1·6 ± 0·8 days, respectively ( P <  0·005). No differences in bleeding events and no transfusion reaction were recorded.
Conclusion  The advantage of an extension of platelet storage time beyond day 5 should be balanced against the increased need for platelet transfusions that may occur and the conceivable risk of transfusion failure.     

Comparison of a novel viscous platelet additive solution and plasma: preparation and in vitro storage parameters of buffy-coat-derived platelet concentrates

Background and Objectives  We developed a viscous platelet additive solution (PAS) based on MacoPharma's SSP+ but containing hydroxyethyl starch to address the poor osmotic balance and low yield associated with conventional PAS for the storage of buffy-coat platelet concentrates (PC).
Materials and Methods  Pools of four buffy-coats were made into leucoreduced PCs ( n  = 5) suspended either in plasma or viscous PAS. After determination of platelet recoveries, the PCs were stored under standard conditions. On days 1, 2, 3, 5, 7 and 9, PCs were tested for mean platelet volume, platelet concentration, soluble protein concentration, CD62 expression, platelet morphology, partial pressure of oxygen and partial pressure of carbon dioxide, glucose and lactate concentration, pH, extent of shape change, and hypotonic shock response (HSR).
Results  Platelets were prepared with greater ease using the viscous PAS and had improved platelet yield. PCs stored in either plasma or viscous PAS displayed similar storage characteristics to day 9. On days 7 and 9 of storage, platelets stored in viscous PAS displayed significantly lower ( P <  0·05) CD62 expression and higher HSR scores than those stored in plasma.
Conclusion  Alteration of the viscosity of PAS improves platelet recovery during processing and may prolong platelet quality at the later stages of storage.     

In vitro storage characteristics of platelet concentrates suspended in 70% SSP+(TM) additive solution versus plasma over a 14-day storage period

Background and Objectives The non‐paired two‐arm study compared the in vitro storage characteristics of platelets suspended as concentrates in either 100% plasma or a mixture of additive solution (SSP+?, MacoPharma, Mouveaux, France) and autologous plasma in a 70:30 ratio over a 14‐day storage period. Materials and Methods The buffy coat‐derived pooled platelet concentrates were sampled on days 1, 2, 3, 6, 8, 10 and 14 and tests performed to determine platelet morphology, function, metabolism, activation and apoptosis‐like activity. Results Swirling remained strong (score = 3) in SSP+?, whilst scores of 1 and 0 were noted for plasma units by end of storage. In contrast to units in plasma, pH levels remained above seven in SSP+? units, increasing after day 10. Percent positive expression of CD62P was similar in both groups on day 1 (median of 54% and 56% for plasma (n = 13) and SSP+? (n = 12), respectively), with SSP+? units showing a more moderate increase in activation after day 10. A progressive decrease in mitochondrial membrane potential was evident in both groups from day 1, whilst annexin V binding was relatively stable from days 1 to 3, with median values remaining below 6%. Subsequent to this, the percentage of platelets binding annexin V increased to approximately 30% by day 14. Conclusion Platelets suspended in a medium of 70:30 SSP+? to plasma ratio performed at least as well as platelets in 100% autologous plasma for up to 10 days of storage. Further, results are suggestive of an apoptosis‐like process being involved in the platelet storage lesion.     

In vitro assessment of platelet storage lesion in leucoreduced random donor platelet concentrates

Background

Currently platelet concentrates (PC) are collected using different synthetic materials and different centrifugation/leucocyte-removal processes. Upon exposure to artificial surfaces and high centrifugation forces, blood cells can undergo various levels of stress-induced, cellular activation/fragmentation and release reactions which may not only influence the extent of the platelet storage lesion but may also contribute to poor clinical effectiveness of the PC and transfusion reactions.

Materials and methods.

An array of assays, used for quality control of PC, was performed in two different groups of PC prepared from random donor plasma on days 1, 3 and 5 of storage. The group 1 PC were not leucoreduced while the group 2 PC underwent prestorage leucoreduction using a PL50E filter. As current recommendations for the evaluation of PC include the measurement of platelet activation, in this study CD62P on platelet membrane was measured. Furthermore, in vitro studies indicate that sHLA antigens may modulate immune competent cell function so, the presence of sHLA-1 in blood components is considered a marker of immunological reactivity and this, too, was measured.

Results

The levels of CD62P and sHLA-1 were significantly lower in leucoreduced PC than in non-leucoreduced ones. However, the overall rate of increase of sHLA-1 during storage was faster in the leucoreduced group of PC. No significant differences were detected regarding other assays of quality.

Conclusion

Based on our findings, leucoreduced PC differ from non-leucoreduced ones in terms of some specific markers such as CD62P as a marker of platelet activation and sHLA-1 as a marker of immunological reactivity. Pre-storage leucofiltration, followed by storage in currently used plastic bags is a safe procedure for PC for up to 5 days. The available leucoreduction technologies are not, however, sufficiently robust to completely abrogate transfusions reactions, and improvements are required to reach the goal of optimised yield and minimal transfusion reactions with platelet therapy.     

Platelet activation at the onset of human endotoxemia is undetectable in vivo

Infection induces platelet activation and consumption, which leads to thrombocytopenia, enhances microvascular thrombosis, impairs microcirculation and eventually triggers disseminated intravascular coagulation (DIC). It is well characterized that endotoxemia results in a pro-inflammatory and pro-coagulatory state, which favors platelet activation. However the early, direct effects of endotoxemia on platelets have not been investigated so far. Therefore we aimed to determine the early effects of the endotoxin lipopolysaccharide (LPS) on platelet function in vivo. In a human endotoxemia model, 15 healthy volunteers were stimulated with LPS (2 ng/kg). Blood was drawn before, 10, 30 and 60 min after LPS challenge and platelet activation analyzed by flow cytometry (GPIIb/IIIa activation, surface CD62P and CD40L, intraplatelet reactive oxygen formation and platelet–leukocyte aggregates) and ELISA (sCD40L, sCD62P and CXCL4). In parallel, blood samples and platelets were spiked with LPS (50 pg/ml) in vitro and monitored over 60 min for the same platelet activation markers.

In vitro platelet stimulation with LPS activated platelets independent of the presence of leukocytes and enhanced their adhesion to endothelial cells. In contrast, in vivo no increase in GPIIb/IIIa activation or surface expression of CD62P was observed. However, endotoxemia resulted in a significant drop in platelet count and elevated the plasma CXCL4 levels already 10 min after the LPS challenge.

These data indicate that LPS rapidly activates platelets, leading to α-granule release and endothelial adhesion. This might explain the drop in platelet count observed at the onset of endotoxemia.     

Correlation of in vitro platelet quality measurements with in vivo platelet viability in human subjects

BACKGROUND AND OBJECTIVES: Changes in in vitro platelet quality parameters during platelet storage are associated with a decrease of in vivo platelet viability after platelet transfusion. Many attempts have been made to identify the most predictable in vitro parameters for in vivo performance. We used a riboflavin-based ultraviolet (UV) light treatment process designed to inactivate pathogens and white blood cell (WBC) contaminants in blood products as a model system in which to study the correlation of in vitro cell quality with in vivo viability. MATERIALS AND METHODS: Platelet products (n = 18) were collected by a standard Trima apheresis procedure and treated with one of three dose levels of UV light (0, 7.2 or 12.4 J/ml) in the presence of 50 microm riboflavin. Lactate production, glucose consumption and P-selectin expression, pH, pCO(2), pO(2), hypotonic shock response and swirl were measured during 5 days of platelet storage post-UV/RB treatment. Aliquots of these products were radiolabelled on day 5 of storage and were subsequently used to determine platelet recovery and survival time in autologous subjects. RESULTS: The responses of in vitro cell quality were observed to occur in a UV dose-dependent manner. Lactate production and pH were identified as the parameters most strongly correlated with platelet in vivo recovery, which ranged from 5 to 82%. The correlation coefficients (r) for lactate production and pH with in vivo recovery in human subjects were 0.9090 and 0.8831 with P-values of 0.007 and 0.031, respectively. Lactate production and pH were also found to be correlated with platelet survival time, with correlation coefficients of 0.8063 and 0.8384 (the P values were 0.01 and 0.001, respectively). CONCLUSIONS: Using conditions of riboflavin-based UV light treatment, lactate production and pH were identified as having the highest correlations with recovery and survival of radiolabelled platelets in healthy subjects.     

Storage of Buffy Coat Preparations at 22°C in Plastic Containers with Different Gas Permeability

Background: Buffy coats (BCs) are used as an alternative to platelet-rich plasma in the preparation of platelet concentrates (PCs). For this purpose the BCs have to be stored for some time at 20–24°C which implies cellular metabolic activity. However, little information is available concerning the effects of a number of factors which may influence the suitability of the preparation as the source of PC. Study design and methods: We studied the effects on BCs of a high and low gas permeability of the wall of the plastic containers, PL2209 and PL146, respectively, mixing versus non-mixing during storage for 48 h at 22°C, and two types of anticoagulant solutions, CPD and half strength citrate CPD (0.5CPD). The buffy coats were prepared by the bottom and top technique. The median values of volume and haematrocrit were 58–64 ml and 39–45%, respectively. A total of 48 BCs were tested. Blood gases, pH, bicarbonate concentration and haemolysis were determined in the blood mixtures and β-thromboglobulin (β-TG), lactate dehydrogenase (LDH), complement factor 3a, and elastase in the extracellular fluid. Results: The pH decreased in all units but to a lesser extent in PL2209 containers than in PL146 units. In the former the pCO₂ decreased slowly in contrast to the latter where it increased by about 50%. Mixing during storage increased the pH and decreased the pCO₂ in 0.5CPD-PL146 and CPD-PL2209 units, as compared to resting, while no effects of mixing were observed in the other groups. The pO₂ decreased to low levels in PL146 units. The haemolysis and LDH release were higher in mixed than in unmixed units. The initial β-TG levels were lowest in 0.5CPD-PL146 units which also had the lowest 24-hour levels. The release of β-TG during storage was smallest in CPD resting units. The elastase release was significantly higher in 0.5CPD than in CPD units already from the beginning of storage and increased during storage at about the same rate irrespective of mixing. The C3a levels were higher in 0.5CPD-PL2209 units than in the other units at 2 h. Storage for 24 h caused an increase by 2–3 times of the original level without any clear relation to storage conditions. Conclusions: In BC units accumulation of CO₂ occurs in containers with low gas permeability. These also show the most rapid pH decrease during storage. Prolonged holding of BCs puts extra emphasis on the need of satisfactory gas permeability of the container for platelet storage in BC-derived PCs. Continuous mixing causes red cell damage and does not seem to have any clear benefit. The release of granulocyte elastase was higher in 0.5CPD than in CPD units but there was no indication of an associated increase in platelet activation. Summary: Study of buffy coats stored in various media and containers at 22°C suggests that it is better to restrict storage to 24 h or less to avoid activation or other deleterious effects on the platelets.     

Prolonged Holding of Whole Blood at 22 °C Does Not Increase Activation in Platelet Concentrates

Objectives: Platelets prepared after holding of whole blood overnight at 22 °C have a well-preserved metabolism. However, the possibility that such prolonged incubation with active granulocytes may increase platelet activation has not been fully tested. Methods: We investigated this possibility by flow cytometric analysis of membrane glycoproteins (GPs) Ib and IIb/IIIa and the activation markers CD62P and CD63 in platelet concentrates (PCs) prepared from whole blood that was held for either 6 h without cooling plates (n = 20) or for 24 h on cooling plates of 1,4-butanediol (n = 20). PCs were prepared by the platelet-rich plasma method and analyzed on the second storage day. Results: Platelet yield and aggregation response to ristocetin, collagen and epinephrine + ADP were similar in both types of PCs, as was the mean fluorescence intensity for GPs Ib and IIb/IIIa. PCs prepared by the overnight-hold method did not differ from those obtained 6 h after collection in the percentage of platelets expressing CD62P (12.3±6.2% vs. 14.1±4.0%; p > 0.1) or CD63 (9.8±6.4% vs. 8.8±3.6%; p > 0.1). Conclusion: Prolonged holding of whole blood at 22 °C prior to component preparation does not increase the level of platelet activation.