Room Temperature-stored Platelets Research Articles

Preservation of recipient plasma sphingosine-1-phosphate levels reduces transfusion-related acute lung injury.

Cold-stored (CS) platelets are once again being reintroduced for clinical use. Transfused CS platelets offer benefits over room temperature-stored (RTS) platelets such as increased hemostatic effects and prolongation of shelf-life. Despite these advantages little is known about their association with transfusion-related acute lung injury (TRALI). TRALI is associated with prolonged storage of RTS platelets and has a mortality of >15%. Determining the safety of CS platelets is important considering their proposed use in TRALI-vulnerable populations with inflammation such as surgical patients or patients with trauma. Donor platelet-derived ceramide causes TRALI, whereas donor platelet sphingosine-1-phosphate (S1P) is barrier protective. Females have higher plasma levels of S1P than males. Cold temperatures increase S1P levels in cells. Therefore, we hypothesized that female (donors or recipients) and/or CS platelets would decrease TRALI. To test this, we compared how male and female donor and recipient allogeneic platelet transfusions of CS (4°C) versus RTS (23°C) platelets stored for 5 days influence murine TRALI. Transfusion of CS platelets significantly reduced recipient lung tissue wet-to-dry ratios, bronchoalveolar lavage total protein, lung tissue myeloperoxidase enzyme activity, histological lung injury scores, and increased plasma sphingosine-1-phosphate (S1P) levels compared with RTS platelet transfusions. Female as opposed to male recipients had less TRALI and higher plasma S1P levels. Female donor mouse platelets had higher S1P levels than males. Mouse and human CS platelets had increased S1P levels compared with RTS platelets. Higher recipient plasma S1P levels appear protective considering females, and males receiving platelets from females or male CS platelets had less TRALI.NEW & NOTEWORTHY Transfusion-related acute lung injury (TRALI) though relatively rare represents a severe lung injury. The sphingolipid sphingosine-1-phosphate (S1P) regulates the severity of platelet-mediated TRALI. Female platelet transfusion recipient plasmas or stored platelets from female donors have higher S1P levels than males, which reduces TRALI. Cold storage of murine platelets preserves platelet-S1P, which reduces TRALI in platelet-transfused recipients.

The impact of donor biological variation on the quality and function of cold-stored platelets.

Room temperature-stored platelets (RTPs) maximize platelet viability but limit shelf life. The aims of this study were to investigate the impact of donor variability on cold-stored platelets (CSPs) and RTP, to determine whether RTP quality markers are appropriate for CSP. Double platelet donations (n = 10) were collected from consented regular male donors stored in 100% plasma. A full blood count, donor age, weight, height and body mass index (BMI) were collected at the time of donation. Platelet donations were split equally into two bags, and assigned to non-agitated CSP or agitated RTP. The quality and function of platelets were assessed throughout the standard 7 days of storage and at expiry (day 8). Non-parametric statistical analyses were used to analyse results given the small sample size. As expected, there were significant differences between CSP and RTP throughout storage including a reduction in CSP concentration as well as a loss of swirling. Furthermore, a significant increase in CSP exhibiting activation and apoptotic markers was observed. Platelet concentrations were further impacted by donor BMI, and donors with the highest BMI (>29) had the lowest platelet concentration and activation response at the end of CSP storage. Platelet quality and functionality play a vital role in transfusion outcomes; however, blood components are inherently variable. This study demonstrated, for the first time, the specific impact of donor BMI on CSP quality and function and highlights the requirement for novel quality markers for assessing CSPs.

Cold-stored Platelets for Bleeding Patients: Comment.

Cold-stored Platelets for Bleeding Patients: Comment.

The haemostatic effect of deep-frozen platelets versus room temperature-stored platelets in the treatment of surgical bleeding: MAFOD—study protocol for a randomized controlled non-inferiority trial

BackgroundThe Netherlands Armed Forces have been successfully using deep-frozen (− 80 °C) thrombocyte concentrate (DTC) for the treatment of (massive) bleeding trauma patients in austere environments since 2001. However, high-quality evidence for the effectiveness and safety of DTCs is currently lacking. Therefore, the MAssive transfusion of Frozen bloOD (MAFOD) trial is designed to compare the haemostatic effect of DTCs versus room temperature-stored platelets (RSP) in the treatment of surgical bleeding.MethodsThe MAFOD trial is a single-blinded, randomized controlled non-inferiority trial and will be conducted in three level 1 trauma centres in The Netherlands. Patients 12 years or older, alive at hospital presentation, requiring a massive transfusion including platelets and with signed (deferred) consent will be included. The primary outcome is the percentage of patients that have achieved haemostasis within 6 h and show signs of life. Haemostasis is defined as the time in minutes from arrival to the time of the last blood component transfusion (plasma/platelets or red blood cells), followed by a 2-h transfusion-free period. This is the first randomized controlled study investigating DTCs in trauma and vascular surgical bleeding.DiscussionThe hypothesis is that the percentage of patients that will achieve haemostasis in the DTC group is at least equal to the RSP group (85%). With a power of 80%, a significance level of 5% and a non-inferiority limit of 15%, a total of 71 patients in each arm are required, thus resulting in a total of 158 patients, including a 10% refusal rate. The data collected during the study could help improve the use of platelets during resuscitation management. If proven non-inferior in civilian settings, frozen platelets may be used in the future to optimize logistics and improve platelet availability in rural or remote areas for the treatment of (massive) bleeding trauma patients in civilian settings.Trial registrationClinicalTrials.gov NCT05502809. Registered on 16 August 2022.

Cold-stored platelet function is not significantly altered by agitation or manual mixing.

Cold storage of platelets (CS-PLT), results in better maintained hemostatic function compared to room-temperature stored platelets (RT-PLT), leading to increased interest and use of CS-PLT for actively bleeding patients. However, questions remain on best storage practices for CS-PLT, as agitation of CS-PLT is optional per the United States Food and Drug Administration. CS-PLT storage and handling protocols needed to be determined prior to upcoming clinical trials, and blood banking standard operating procedures need to be updated accordingly for the release of units due to potentially modified aggregate morphology without agitation. We visually assessed aggregate formation, then measured surface receptor expression (GPVI, CD42b (GPIbα), CD49 (GPIa/ITGA2), CD41/61 (ITGA2B/ITGB3; GPIIB/GPIIIA; PACI), CD62P, CD63, HLAI), thrombin generation, aggregation (collagen, adenosine diphosphate [ADP], and epinephrine activation), and viscoelastic function (ExTEM, FibTEM) in CS-PLT (Trima collection, 100% plasma) stored for 21 days either with or without agitation (Phase 1, n=10 donor-paired units) and then without agitation with or without daily manual mixing to minimize aggregate formation and reduce potential effects of sedimentation (Phase 2, n=10 donor-paired units). Agitation resulted in macroaggregate formation, whereas no agitation caused film-like sediment. We found no substantial differences in CS-PLT function between storage conditions, as surface receptor expression, thrombin generation, aggregation, and clot formation were relatively similar between intra-Phase storage conditions. Storage duration and not condition impacted phenotype and function. CS-PLT can be stored with or without agitation, and with or without daily mixing and standard metrics of hemostatic function will not be significantly altered.

Divalent magnesium restores cytoskeletal storage lesions in cold-stored platelet concentrates

Cold storage of platelet concentrates (PC) has become attractive due to the reduced risk of bacterial proliferation, but in vivo circulation time of cold-stored platelets is reduced. Ca2+ release from storage organelles and higher activity of Ca2+ pumps at temperatures < 15 °C triggers cytoskeleton changes. This is suppressed by Mg2+ addition, avoiding a shift in Ca2+ hemostasis and cytoskeletal alterations. We report on the impact of 2–10 mM Mg2+ on cytoskeleton alterations of platelets from PC stored at room temperature (RT) or 4 °C in additive solution (PAS), 30% plasma. Deformation of platelets was assessed by real-time deformability cytometry (RT-DC), a method for biomechanical cell characterization. Deformation was strongly affected by storage at 4 °C and preserved by Mg2+ addition ≥ 4 mM Mg2+ (mean ± SD of median deformation 4 °C vs. 4 °C + 10 mM Mg2+ 0.073 ± 0.021 vs. 0.118 ± 0.023, p < 0.01; n = 6, day 7). These results were confirmed by immunofluorescence microscopy, showing that Mg2+ ≥ 4 mM prevents 4 °C storage induced cytoskeletal structure lesion. Standard in vitro platelet function tests showed minor differences between RT and cold-stored platelets. Hypotonic shock response was not significantly different between RT stored (56.38 ± 29.36%) and cold-stored platelets with (55.22 ± 11.16%) or without magnesium (45.65 ± 11.59%; p = 0.042, all n = 6, day 1). CD62P expression and platelet aggregation response were similar between RT and 4 °C stored platelets, with minor changes in the presence of higher Mg2+ concentrations. In conclusion, increasing Mg2+ up to 10 mM in PAS counteracts 4 °C storage lesions in platelets, maintains platelet cytoskeletal integrity and biomechanical properties comparable to RT stored platelets.

Effect of bedside filtration on aggregates from cold-stored whole blood-derived platelet-rich plasma and apheresis platelet concentrates.

The current approach to manufacture cold-stored platelets (CSP) replicates that of room temperature-stored platelets (RSP). However, this production method is associated with aggregate formation in CSP, a major pitfall that leads to significant wastage. We hypothesized that isolating platelets from whole blood as platelet-rich plasma (PRP) and storing them at a lower concentration reduces aggregates and that conventional bedside transfusion filtration removes CSP aggregates. We collected platelets from healthy humans by apheresis (AP) and by phlebotomy, from which we generated platelet-rich plasma (PRP). We split each AP and PRP platelets into two equal aliquots, storing one at 22°C (RT-PRP and RT-AP) and the other at 4°C (4C-PRP and 4C-AP). We evaluated platelets on day 0 and day 7 of storage. After storage, we measured platelet counts, aggregates, and other key characteristics before and after filtration by a bedside filter. After storage, the 4C-AP platelet counts decreased significantly. 4C-PRP preserved glucose better and prevented a significant increase in lactate contrary to 4C-AP. Filtration led to significantly lower platelet counts in both 4C-PRP and 4C-AP but not in their RT counterparts. Post filtration, we observed 50% fewer aggregates only in 4C-AP, whereas 4C-PRP showed an unexpected but significant increase in aggregates. Testing confirmed activation during storage but filtration did not further activate platelets. We provide evidence that 4C-PRP is an alternative to 4C-AP and that bedside filters reduce aggregates from 4C-AP. Further studies are needed to evaluate the hemostatic potential of 4C-PRP and the management of aggregates.

Donor and Storage Duration Impact Cold-Stored Platelet Function More Than Handling Protocols

Cold storage of platelets has the potential to mitigate the logistical and financial burdens associated with platelet inventory management. In addition, cold-stored platelets (CS-PLT, stored at 2-6°C) have better preserved hemostatic function than room temperature (RT)-stored platelets (RT-PLT), suggesting that CS-PLT may provide improved hemostatic resuscitation in actively bleeding patients as compared to RT-PLT when transfused. With CS-PLT clinical use on the rise, questions regarding CS-PLT inventory management have been raised - specifically how should CS-PLT be stored and handled over the course of storage (after collection and prior to transfusion). RT-PLT are currently stored flat on specialized porous racks and agitated at 18-22°C, which allows for oxygenation. These products are stored out to 5 days, with 7 days allowed for large volume delayed sampling collections. In contrast, refrigerated blood products (i.e., packed red blood cells and whole blood) are stored vertically in pull out drawers, with the goal of reducing harmful oxidizing damage to red blood cells. Notably, refrigeration is known to induce aggregation of platelets, a feature historically associated with bacterial contamination in RT-PLT. In order to diminish concerns regarding aggregate formation, it may be beneficial to massage CS-PLT briefly each day to reduce aggregate formation, yet how this may impact hemostatic function is unknown. To this end, optimal storage and handling conditions for CS-PLT remain to be determined.The objective of our study was to measure CS-PLT hemostatic function in response to two major storage and handling variables: agitation and massage to reduce aggregation in the bag. Single donor apheresis platelet units (single Trima collection in plasma, split equally into two bags to control for donor variability; n=20 donors in 40 bags) were purchased from our regional blood center and delivered to our lab on day of collection. Upon arrival, units were spiked and sampled under sterile conditions for baseline (day 0) profiling, assigned to one of the study groups, and stored accordingly. Our study groups were as follows: “Agitated” (n=10 units), “Not Agitated” (n=10 units, paired with Agitated donors), “Massaged Daily”, (massaged 60s daily, n=10 units), and “Massaged at Sampling” (massaged only at sampling, n=10 units, paired with Massaged Daily donors). “Agitated” platelet units were stored in a custom refrigerated shaker (courtesy Helmer Scientific). For the other groups, platelet units were stored in a walk-in cold room. All units were stored on the same style perforated agitator rack, with agitation either powered on or off as assigned. Units from all groups were sampled under sterile conditions at days 2, 5, 7, 14, and 21 of storage, using 8 mL draws at each time point to ensure equal volume removal over the course of storage. Hemostatic function was assayed using light transmission aggregometry (LTA; ADP, collagen, epinephrine agonists), rotational thromboelastometry (ROTEM; ExTEM, InTEM agonists), and thrombin generation in response to 5 pM tissue factor. Platelet counts (x10 3/µL) were obtained using a hematology analyzer.During the first week of storage, there were no significant differences in the hemostatic profiles between study groups. While platelet counts, endogenous thrombin potential, and ROTEM clot formation time and maximum clot firmness were fairly stable over the first 7 days of storage across all groups, there was a > 50% reduction from baseline in aggregation responses to both ADP and collagen across all 4 groups, suggesting that platelet aggregation, as detected by LTA, may not be the best representative of CS-PLT function. By day 21 of storage, there was a robust increase in endogenous thrombin potential in all study groups when compared to baseline (Agitated, 22%; Not Agitated, 70%; Massaged Daily, 51%; Massaged at Sampling, 47%). Despite this increase in thrombin generation, after extrinsic activation day 21 CS-PLT in all groups had two-fold increased clot formation times compared to baseline (Agitated, 185%; Not Agitated, 223%; Massaged Daily, 236%; Massaged at Sampling, 190%), and reduced maximum clot firmness compared to baseline (Agitated, -44%; Not Agitated, -36%; Massaged Daily, -54%; Massaged at Sampling, -43%). These data suggest that storage duration, and not agitation nor massaging to reduce aggregates, has the most impact on CS-PLT hemostatic function. DisclosuresSpinella: Cerus Corporation: Consultancy, Research Funding; Secure Transfusion Services: Current Employment, Current holder of individual stocks in a privately-held company, Current holder of stock options in a privately-held company.

Storage temperature determines platelet GPVI levels and function in mice and humans

AbstractPlatelets are currently stored at room temperature before transfusion to maximize circulation time. This approach has numerous downsides, including limited storage duration, bacterial growth risk, and increased costs. Cold storage could alleviate these problems. However, the functional consequences of cold exposure for platelets are poorly understood. In the present study, we compared the function of cold-stored platelets (CSP) with that of room temperature–stored platelets (RSP) in vitro, in vivo, and posttransfusion. CSP formed larger aggregates under in vitro shear while generating similar contractile forces compared with RSP. We found significantly reduced glycoprotein VI (GPVI) levels after cold exposure of 5 to 7 days. After transfusion into humans, CSP were mostly equivalent to RSP, yet aggregated significantly less to the GPVI agonist collagen. In a mouse model of platelet transfusion, we found a significantly lower response to the GPVI-dependent agonist convulxin and significantly lower GPVI levels on the surface of transfused platelets after cold storage. In summary, our data support an immediate but short-lived benefit of cold storage and highlight the need for thorough investigations of CSP. This trial was registered at www.clinicaltrials.gov as #NCT03787927.

Platelets Feeding Bacteria with Lactate during Room Temperature Storage: Mitigated By Refrigeration

Introduction: Platelet transfusion related sepsis is a serious concern limiting platelet storage time to 5 days. Room temperature (RT) storage of platelets increases the risk of bacterial growth and necessitates implementation of bacterial contamination monitoring. Cold storage of platelets represents an attractive alternative for improving platelet safety. In this study, we assessed bacterial growth in platelets stored either at room temperature (22oC) or refrigerated (4oC).Methods: Apheresis platelets in plasma (PLT) were obtained from healthy donors using the Terumo Trima Accel Automated Blood Collection System (Terumo BCT). Platelet poor plasma (PPP) was obtained from PLT aliquots centrifuged twice at 2,500 x g for 5 min. In some experiments aliquots of PPP were supplemented with 5 to 20 mM lactic acid. Aliquots of PLT or PPP were transferred to pH SAFE minibags (Blood Cell Storage, Inc) and inoculated with Acinetobacter baumannii strains Ci77 or Ci79, Escherichia coli, Pseudomonas aeruginosa, or PBS (uninfected control). Minibag aliquots stored at RT were agitated using an orbital shaker set to 60 rpm while refrigerated aliquots were stored under static conditions. Bacterial growth was monitored daily through dilution plating. Lactate levels in PLT aliquots were assessed by iSTAT (Abbott) using CG4+ test cartridges. Platelet activation and aggregation were assessed on days 0, 1, 3, and 5 by flow cytometry and Multiplate platelet aggregometry respectively.Results: Growth of A. baumannii progressed exponentially over the first 3 days post-collection in PLT aliquots stored at RT. However, growth was significantly (p < 0.05) reduced in PPP units. Growth of E. coli and P. aeruginosa, both of which are capable of utilizing glucose present in the anticoagulant used, grew at significantly faster rates and did not seem to be dependent on metabolically active PLTs. Lactate levels were assessed in PLT units and found to mirror growth of A. baumannii . Furthermore, addition of ≥ 15 mM lactic acid to RT stored PPP restored A. baumannii growth. Although lactate levels did not appear to contribute to growth of E. coli, lactate levels began to decrease in PLT units contaminated with the bacterium at a rapid rate after day 3. Lactate levels in units contaminated with P. aeruginosa increased throughout the observation window. Bacterial growth remained static throughout under all treatment conditions stored refrigerated and lactate levels were reduced. PLT also appeared to retain function throughout cold storage irrespective of bacterial contamination while RT storage resulted in a decrease in aggregation over time which was exacerbated by bacterial contamination.Conclusions: Bacterial growth remained static throughout under cold storage. Additionally, PLT lactate production was increased at RT and correlated with increased growth of A. baumannii . Growth of E. coli and P. aerugionsa appeared to proceed independently of lactate production or metabolically active PLTs. However, E. coli did appear to utilize lactate as a carbon source at later time points. These data demonstrate that bacterial growth can be controlled through refrigeration without loss of function and RT stored platelets may potentiate growth of certain bacterial strains through accelerated metabolism relative to cold storage. DisclosuresNo relevant conflicts of interest to declare.

Calcium Ion Chelation Preserves Platelet Function During Cold Storage.

Platelet transfusion is a life-saving therapy to prevent or treat bleeding in patients with thrombocytopenia or platelet dysfunction. However, for >6 decades, safe and effective strategies for platelet storage have been an impediment to widespread use of platelet transfusion. Refrigerated platelets are cleared rapidly from circulation, precluding cold storage of platelets for transfusion. Consequently, platelets are stored at room temperature with an upper limit of 5 days due to risks of bacterial contamination and loss of platelet function. This practice severely limits platelet availability for transfusion. This study is to identify the mechanism of platelet clearance after cold storage and develop a method for platelet cold storage. Approach and Results: We found that rapid clearance of cold-stored platelets was largely due to integrin activation and apoptosis. Deficiency of integrin β3 or caspase-3 prolonged cold-stored platelets in circulation. Pretreatment of platelets with EGTA, a cell impermeable calcium ion chelator, reversely inhibited cold storage-induced platelet activation and consequently prolonged circulation of cold-stored platelets. Moreover, transfusion of EGTA-treated, cold-stored platelets, but not room temperature-stored platelets, into the mice deficient in glycoprotein Ibα significantly shortened tail-bleeding times and diminished blood loss. Integrin activation and apoptosis is the underlying mechanism of rapid clearance of platelets after cold storage. Addition of a cell impermeable calcium ion chelator to platelet products is potentially a simple and effective method to enable cold storage of platelets for transfusion.

Use of Specialized Pro-Resolving Mediators to Alleviate Cold Platelet Storage Lesion.

Cold-stored platelets are an attractive option for treatment of actively bleeding patients due to a reduced risk of septic complications and preserved hemostatic function compared to conventional room temperature-stored platelets. However, refrigeration causes increased platelet activation and aggregate formation. Specialized pro-resolving mediators (SPMs), cell signaling mediators biosynthesized from essential fatty acids, have been shown to modulate platelet function and activation. In this study, we sought to determine if SPMs could be used to inhibit cold-stored platelet activation. Platelets were collected from healthy donors (n = 4-7) and treated with SPMs (resolvin E1 [RvE1], maresin 1 [MaR1], and resolvin D2 [RvD2]) or vehicle (VEH; 0.1% EtOH). Platelets were stored without agitation in the cold and assayed on Days 0 and 7 of storage for platelet activation levels using flow cytometry, platelet count, aggregation response using impedance aggregometry, and nucleotide content using mass spectrometry. Compared to VEH, SPM treatment inhibited GPIb shedding (all compounds), significantly reduced both PS exposure and activation of GPIIb/IIIa receptor (RvD2, MaR1), and preserved aggregation response to TRAP (RvD2, MaR1) after 7 days of storage. Similar to untreated cold-stored platelets, SPM-treated samples did not preserve platelet counts or block the release of P-Selectin. Nucleotide content was unaffected by SPM treatment in cold-stored platelets. SPM treatment, particularly Mar1 and RvD2, led to reduced platelet activation and preserved platelet function after 7 days of storage in the cold. Future work is warranted to better elucidate the mechanism of action of SPMs on cold platelet function and activation.

Evaluation of Efficacy and Safety of Cold-Stored Platelets in Healthy Human Subjects Treated with Dual Antiplatelet Therapy

Background: Platelets are currently stored at 22°C (room temperature-stored) for clinical purposes. This approach has numerous downsides including limited storage time due to risk of bacterial growth, and increased costs caused by bacterial testing or pathogen reduction. Platelets stored at 4ºC (cold-stored) were the standard of care in the 1960s and 70s, and while they generally perform better than room temperature-stored platelets in in vitro assays, functional studies in vivo have yielded contradictory results. Methods: Eight healthy human participants were included in the analysis of this randomized, cross-over study. A double unit of apheresis platelets was collected from each subject for autologous transfusion. Platelets were stored for 5 days at either 4ºC, or 22ºC, based on randomization. On day 5, platelets were transfused 12-24 hours after participants received a loading dose of acetylsalicylic acid and clopidogrel. An array of platelet function testing was done at baseline, after loading dose, and after transfusion (1h, 4h, 24h). The first round was followed by a wash-out period of at least 10 days, and a second sequence of collection, antiplatelet loading dose, and transfusion with platelets stored under alternate conditions from the first sequence. The primary endpoint was defined as post-transfusion platelet αIIbβ3 integrin activation in response to agonists for pathways inhibited by dual antiplatelet therapy, measured by the VerifyNOW assay. Secondary endpoints included platelet function testing by bleeding time, VASP-phosphorylation, light transmission aggregometry, and flow cytometric analysis of platelet αIIbβ3 integrin activation and α-granule secretion. Results: All cold-stored platelet units passed quality control analyses, and no platelet aggregates were observed at the end of the storage period. One room temperature-stored unit had to be discarded due to quality control failure. Transfusion of a double unit with 5 day cold-stored platelets was tolerated well by all recipients. The absolute number of transfused platelets did not differ significantly between the cold-storage arm and the room temperature storage arm. The corrected count increment did not differ significantly at 1h after transfusion, but was significantly lower in the cold-stored group at the 4h, and 24h time points. The primary endpoint, platelet function testing by VerifyNOW, showed reversal of the effect of acetylsalicylic acid with both products at 1h and 4h post transfusion, but platelet inhibition re-appeared after 24h post transfusion in the cold-storage arm, presumably due to accelerated clearance of cold-stored platelets. No significant differences were observed between the two products in the VerifyNOW assay for clopidogrel. The bleeding time (a secondary endpoint) did not differ significantly at any time point between the cold-stored and room temperature-stored transfusion group. However, the bleeding time improved or remained unchanged in all recipients transfused with room temperature-stored platelets, whereas, transfusion of cold-stored platelets caused prolongation of the bleeding time in 50% of recipients. Surprisingly, platelets isolated from recipients of room temperature-stored platelets aggregated significantly better in response to collagen compared to platelets isolated from recipients of cold-stored platelets at 1h and 4h post transfusion. Other secondary endpoints, including VASP-phosphorylation, αIIbβ3 integrin activation and α-granule secretion by flow cytometry, and platelet aggregation in response to arachidonic acid and ADP did not differ significantly between the two treatment groups. Conclusion: We report the first safety and efficacy data of 5 day cold stored platelets in plasma. Unexpectedly, some endpoints demonstrate agonist- and assay-dependent inferiority of cold-stored platelets at early and late time points. Further studies are needed to determine the maximum storage time, and the efficacy of cold-stored platelets in actively bleeding patients and patients with platelet dysfunction. Disclosures No relevant conflicts of interest to declare.

Cold-stored whole blood platelet function is preserved in injured children with hemorrhagic shock.

Recent data demonstrate the safety of uncrossmatched cold-stored whole blood (WB) transfusion in pediatric trauma patients. The hemostatic capabilities of platelets within the cold-stored WB unit have been demonstrated via in vitro studies and animal models. However, platelet function has not been evaluated in pediatric recipients of cold-stored WB transfusions. Injured children, 2 years or older and 10 kg or greater with hemorrhagic shock received up to 30 mL/kg of cold-stored, low titer (<50) anti-A and -B, leukoreduced, group O- WB during their initial resuscitation. Patients were included if (1) they received WB and no conventional platelets, and (2) platelet count and thromboelastography maximum amplitude were measured both before and after transfusion. These data and relevant clinical outcomes (mortality, intensive care unit length of stay [LOS], hospital LOS and ventilator days) were compared to a historical cohort of pediatric trauma patients who received uncrossmatched red blood cells (RBC) and conventional room temperature platelets. Twenty-two children were included in the study; 14 in the component cohort versus 8 in the WB cohort. Neither posttransfusion platelet count (129 × 109/L vs. 135 × 109/L) nor function (thromboelastography maximum amplitude, 59.5 mm vs. 60.2 mm) differed significantly between children receiving cold-stored platelets within the WB unit versus children who received conventional warm platelets. Median (interquartile range) weight-adjusted platelet transfusion volume in the historical cohort was 4.6 (2.5-7.7) mL/kg vs. 2.4 (1.3-4.0) mL/kg in the WB cohort (p = 0.03). There was no difference between groups in age, race, mechanism of injury, Injury Severity Score, vital signs, and severe traumatic brain injury (TBI). Outcomes, including mortality, intensive care unit LOS, hospital LOS, and ventilator days, were not significantly different between groups. No difference was seen in posttransfusion platelet number or function in severely injured children receiving cold-stored WB platelets as compared to those receiving conventional room temperature-stored platelets. Larger cohorts are required to confirm these findings. Therapeutic, level IV.

Just chill—it's worth it!

Just chill—it's worth it!

Platelet Lactate Production during Room Temperature Storage Promotes Bacterial Growth

Introduction: Transfusion related sepsis is a serious concern for both military and civilian trauma centers. The primary source of bacterial contamination associated with such events is room temperature (RT) stored platelets. Due to high risk of bacterial contamination, RT stored platelets may only be administered up to 5 days post-collection. Although approved by the FDA, cold stored platelets may only be administered 3 days post-collection under current regulations. In this study, we utilized the opportunistic pathogen Acinetobacter baumannii - a reported cause of platelet contamination and important hospital acquired infection (HAI) - as a model organism to assess bacterial growth in platelets stored either at room temperature (22oC) or refrigerated (4oC), as well as determine contributors to bacterial growth.Methods: Apheresis platelets in plasma (PLT) were obtained from healthy donors using the Terumo Trima Accel Automated Blood Collection System (Terumo BCT). Platelet poor plasma (PPP) was obtained from PLT aliquots centrifuged twice at 2,500 x g for 5 min. Other PLT aliquots were stimulated with Phorbol 12-myristate 13-acetate (PMA) to generate activated platelets (aPLT). Following activation, aPLTs were pelleted through centrifugation at 2,500 x g for 5 min and the releasate (REL) was transferred to a separate container. Pelleted aPLTs were washed 3 times in sterile PBS and suspended in PPP. In some experiments aliquots of PPP were supplemented with 40 mM lactic acid. Aliquots of PLT, PPP, aPLT, and REL were transferred to pH SAFE minibags (Blood Cell Storage, Inc) and inoculated (a.k.a., contaminated) with A. baumannii clinical isolate Ci79. Minibag aliquots stored at RT were agitated using an orbital shaker set to 60 rpm while refrigerated aliquots were stored under static conditions. Bacterial growth was monitored daily through dilution plating. In some experiments, lactate levels in PLT aliquots were assessed by iSTAT (Abbott) using CG4+ test cartridges.Results: Bacterial growth progressed exponentially over the first 3 days post-collection in PLT aliquots stored at RT. However, growth was significantly (p < 0.05) reduced in PPP units (Fig. 1A). Neither platelet activation status nor released factors appeared to have any effect as no significant differences were observed in bacterial growth between contaminated PLT and aPLT units, nor between contaminated PPP and REL units (Fig. 1A). Growth progressed at a much faster rate and to a greater magnitude in the presence of live platelets (PLT and aPLT), suggesting the contribution of platelet metabolism. Thus, lactate levels were assessed in PLT units and found to mirror bacterial growth (Fig. 1B). Furthermore, addition of lactic acid to RT stored PPP restored bacterial growth (Fig. 1A). Growth remained static throughout under all treatment conditions stored refrigerated.Conclusions: Bacterial growth remained static over the 5 day post-collection observation period during cold storage. Additionally, bacterial growth at RT appeared to be related to increased production of lactate from pyruvate via NAD-dependent lactate dehydrogenase (nLDH) following glycolysis (Fig. 1C). To that end, many bacteria, including A. baumannii,as well as various Staphylococcus spp, and Streptococcus spp, possess genes encoding for NAD-independent lactate dehydrogenases (iLDH) which enable utilization of lactate as a carbon source (Fig. 1D). These data demonstrate that not only can bacterial growth be controlled through refrigeration, but RT stored platelets potentiate bacterial growth through their accelerated metabolism relative to cold storage. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Comprehensive review of platelet storage methods for use in the treatment of active hemorrhage.

This review considers the various methods currently in use, or under investigation, for the storage of platelets intended for use in the treatment of active hemorrhage. The current standard practice of storing platelets at room temperature (RT) (20°C-24°C) optimizes circulating time, but at the expense of hemostatic function and logistical considerations. A number of alternatives are under investigation. Novel storage media additives appear to attenuate the deleterious changes that affect RT stored platelets. Cold storage was originally abandoned due to the poor circulating time of platelets stored at 4°C, but such platelets may actually be more hemostatically effective, with a number of other advantages, compared to RT stored platelets. Periodically re-warming cold stored platelets (temperature cycling, TC) may combine the hemostatic efficacy of cold stored platelets with the longer circulating times of RT storage. Alternatives to liquid storage include cryopreservation (freezing) or lyophilization (freeze-drying). The former has had some limited clinical use and larger clinical trials are underway, while the latter is still in the preclinical stage with promising in vitro and in vivo results. The importance of platelet transfusion in the management of active hemorrhage is now well accepted, so it is timely that platelet storage methods are reviewed with consideration of not only their circulating time, but also their hemostatic efficacy.

Risk and prevention of transfusion-related sepsis.

Transfusion-related sepsis is both the oldest recognized and most common transfusion-associated infectious risk. Despite an increased awareness and recognition of this problem, particularly with room-temperature stored platelets, strategies to prevent or reduce the occurrence of this problem have proved daunting. With the recent FDA approval of culture methods for platelet bacterial testing and the promulgation of accreditation standards by the College of American Pathologists and American Association of Blood Banks to limit and detect platelet bacterial contamination, it is anticipated that the frequency of this problem will now begin to diminish. As methods to reduce and/or inactivate pathogens emerge, it is hoped that transfusion-related sepsis will essentially disappear.

Cold temperatures reduce the sensitivity of stored platelets to disaggregating agents

In this study, we compared the effect of signal transduction inhibitors on fibrinogen binding, aggregation, the activation state of GPIIb-IIIa, and cytosolic calcium levels in cold and room temperature-stored platelets. Cold-stored platelets have a higher sensitivity to agonist-induced aggregation when compared to room temperature-stored platelets. We also found that cold-stored platelets had a significantly higher aggregation response to ADP and epinephrine, while platelets stored at room temperature responded poorly to these agonists (mean values of 61 vs. 18%, n = 14). Four inhibitors were selected to target various signaling pathways. Cold-stored platelets were more resistant to disaggregation by promethazine, prostaglandin D2, yohimbine, and echistatin. The effects of cold temperatures on stored platelets are targeted to activation pathways as there was no spontaneous aggregation or spontaneous fibrinogen binding as measured in this study. PAC-1 binding was not inhibited to the same degree as aggregation or fibrinogen binding responses, suggesting that the disaggregation was not caused by a change in the conformation of GPIIb-IIIa. Cytosolic calcium levels did not decrease in cold-stored platelets after inhibitor addition. The inhibitors are likely acting after the establishment of the GPIIb-IIIa activation state and may affect the post-occupancy signaling by the fibrinogen-occupied integrin. Differences between aggregation and disaggregation responses of cold- and room temperature-stored platelets suggest that cold-stored platelets may have different mechanisms to stabilize platelet aggregates during their formation.

Novel treatment modalities: new platelet preparations and substitutes.

Keywords: platelets; platelet substitutes; platelet haemostatic function; blood substitutes; platelet function