Cold storage of whole blood in an additive solution containing apoptotic and necrotic inhibitors.

BACKGROUND: Whole blood (WB) reigns superior to component therapy for the treatment of hemorrhagic shock on the battlefield. Though cold storage of WB offers a shelf life of 21 to 35 days, storage lesions and the potential for blood wastage remain. Storing WB in an additive solution (AS) containing apoptotic inhibitors may help preserve blood cell viability and improve blood quality over extended cold storage. STUDY DESIGN AND METHODS: Non-leukoreduced WB was obtained from healthy individuals and dosed with: AS, AS+Necrostatin-1 (AS+N1), AS+Boc-D-fmk (AS+B; apoptosis inhibitor), AS+Q-VD-OPh (AS+Q; apoptosis inhibitor), and Control (0.9% saline). Blood bags were kept refrigerated (1°-6°C) for 21 days. Bags were tested on days 0, 7, 14, and 21 for complete blood count, metabolism, clot formation, aggregation function, platelet activation, and red blood cell quality. RESULTS: Platelet count was better preserved in all AS-containing samples. All groups displayed increased glucose consumption and lactate production with storage. Furthermore, all groups displayed a similar decline in clot strength (max amplitude) over the 21-day storage period. Bags that received AS displayed greater preservation of GPIIb expression and lower phosphatidylserine exposure. P-selectin expression was increased in all AS groups. DISCUSSION: Treatment of hemorrhagic shock with WB transfusion is logistically simpler than component therapy. Results from our study suggest that refrigerated WB stored with an AS containing apoptotic and necrotic inhibitors helps better preserve platelet count but does not improve platelet function. The future development of WB ASs is warranted to optimize both platelet quality and hemostatic function.

Metabolic effects of radiation on red blood cells from cold stored whole blood.

BACKGROUND: The risk of military and civilian radiation exposure is increasing, and determining the effects of exposure is a high priority. Irradiation of the nearby blood supply after a nuclear event may impede mobilization of blood products for resuscitation at a time of great need. RBCs are administered to patients with trauma and hemorrhage to transport and deliver oxygen and avoid tissue hypoxia. Here we determine the effects of ionizing radiation on the energy metabolome of RBCs isolated from cold stored whole blood to determine if their stability is compromised by radiation exposure. STUDY DESIGN AND METHODS: Whole blood from healthy volunteers was subjected to 0, 25, or 75 Gy of X-irradiation, and stored at 4°C. RBCs were isolated from stored WB at 0, 1, 7, 14, and 21 days of storage. The levels of extracted Krebs cycle intermediates, nicotinamide adenine dinucleotides, and phosphorylated derivatives of adenosine and guanosine were determined by tandem mass spectroscopy. RESULTS: Irradiation at either 25Gy or 75Gy had no significant effect on any parameter measured compared to control (0Gy). However, there was a significant change over time in storage for ATP, GDP, and guanosine. DISCUSSION: Irradiation at doses up to 75Gy had no effect on the energy metabolome of RBCs prepared from blood stored at 4°C for up to 21 days, suggesting that the RBC energy metabolome is not affected by radiation exposure and the blood can still be used for resuscitation in trauma patients.

Effects of radiation exposure and storage on the energy metabolome of platelets in whole blood.

BACKGROUND: Exposure to radiation through battlefield use of nuclear weapons, terrorist attacks or accidents at nuclear power plants is a current concern for the military. Beyond the risk of exposure to personnel is the intentional or accidental irradiation of our blood banking supply system. It is unknown how large doses of ionizing radiation affect storage of blood and blood products, including platelets. The major function of platelets is clot formation which includes aggregation, shape change, vesicle release, and fibrinogen attachment; these tasks require a significant amount of energy. Here, we determine whether the ionizing radiation effects the energy metabolome of platelets in storage. STUDY DESIGN AND METHODS: Fresh whole blood from healthy volunteers was subjected to 0, 25, or 75Gy of X-irradiation, and stored at 4°C. Platelets were isolated from stored WB at 0, 1, 7, 14, and 21 days of storage. Krebs cycle intermediates, nicotinamide adenine dinucleotides, and the tri-, di, and mono- phosphorylated versions of adenosine and guanosine were extracted and measured by tandem mass spectroscopy. RESULTS: Irradiation at either 25Gy or 75Gy had no significant effect on the amount of any metabolite measured compared to control (0Gy). However, there was a significant fall over time in storage for most of the metabolites measured. DISCUSSION: These data show that irradiation at high doses has no effect on the concentration of the energy metabolome of platelets derived from whole blood stored in 4°C for up to 21 days and suggests that platelets can maintain their metabolome even after radiation exposure.

Refrigerated human mesenchymal stromal cells as an alternative to cryostorage for use in clinical investigation.

BACKGROUND: Mesenchymal stromal cells (MSCs) and other therapeutic cells show efficacy for cardiac damage, neurological disease, chronic lung disease, pediatric graft versus host disease, and several inflammatory conditions. Based on their anti-inflammatory and immune-modulatory activities, responsiveness, and secretion of beneficial factors, cellular therapeutics may provide benefits in acute and chronic traumatic injury. However, the use of live cells presents logistical challenges, especially for military trauma. MSCs are generally shipped and stored frozen but require sterile handling before infusion. This requires skilled personnel and equipment not readily available in a forward medical treatment facility or even a small community hospital. METHODS: Commercial human bone marrow- and adipose-derived MSCs from multiple donors were cultured under standard conditions, harvested and stored at 4°C in solution for up to 21 days. Cell viability, ATP content, apoptosis, proliferation capability, immunomodulation activity, and responsiveness were assessed after different amounts of time. RESULTS: Human MSCs can be stored at 4°C in MSC culture medium for 14 days while maintaining a reasonable level of viability and function. Both viability and function are reduced when MSCs are stored in crystalloid solutions. CONCLUSIONS: This approach makes it feasible to prepare cellular therapeutic agents in a laboratory or commercial facility and ship them under refrigerated conditions. Once they reach their destination, they can be stored at 4°C under conditions similar to blood products. Cells prepared and stored this way could also be used directly with minimal handling, making them more practical for both civilian and military trauma.

Comparison of platelet quality and function across apheresis collection platforms.

BACKGROUND: Platelet concentrates (PLT) can be manufactured using a combination of apheresis collection devices and suspension media (plasma or platelet additive solution (PAS)). It is unclear how platelet quality and hemostatic function differ across the current in-use manufacturing methods in the United States. The objective of this study was therefore to compare baseline function of PLT collected using different apheresis collection platforms and storage media. STUDY DESIGN AND METHODS: PLT were collected at two sites with identical protocols (N = 5 per site, N = 10 total per group) on the MCS® + 9000 (Haemonetics; "MCS"), the Trima Accel® 7 (Terumo; "Trima"), and the Amicus Cell Separator (Fresenius Kabi, "Amicus"). MCS PLT were collected into plasma while Trima and Amicus PLT were collected into plasma or PAS (Trima into Isoplate and Amicus into InterSol; yielding groups "TP", "TI" and "AP", "AI", respectively). PLT units were sampled 1 h after collection and assayed to compare cellular counts, biochemistry, and hemostatic function. RESULTS: Differences in biochemistry were most evident between plasma and PAS groups, as anticipated. MCS and TP had the highest clot strength as assessed by viscoelastometry. AI had the lowest thrombin generation capacity. Both TP and TI had the highest responses on platelet aggregometry. AI had the greatest number of microparticles. DISCUSSION: Platelet quality and function differ among collection platforms at baseline. MCS and Trima platelets overall appear to trend toward higher hemostatic function. Future investigations will assess how these differences change throughout storage, and if these in vitro measures are clinically relevant.

A novel, quantitative clot retraction assay to evaluate platelet function.

Blood platelets are crucial to prevent excessive bleeding following injury to blood vessels. Platelets are crucial for the formation of clots and for clot strength. Platelet activation involves aggregation, attachment to fibrin and clot retraction. Most assays that address platelet function measure platelet aggregation, not clot retraction. Here, we describe a 96-well-based clot retraction assay that requires a relatively short runtime and small sample volume. The assay involves continuous optical density monitoring of platelet-rich plasma that is activated with thrombin. The data can be analyzed using time-series analytical tools to generate quantitative information about different phases of clot formation and clot retraction. The assay demonstrated good repeatability and reproducibility and was robust to different calcium concentrations. Impairment of platelet bioenergetics, actin polymerization, fibrin interaction, and signaling significantly affected clot retraction and was detected and showed good agreement with light transmission aggregometry, suggesting that clot retraction is predictive of platelet function. Using this microplate clot retraction assay, we showed a significant difference in platelets stored in autologous plasma compared with platelet additive solution after 7 days of room temperature storage.

Platelets are cell fragments in the blood that are necessary for clot formation. They are crucial to preventing excessive bleeding following trauma. To form clots, platelets clump (aggregate) and attach to fibrin protein and cells inside the blood vessels to form strong web-like structures. Platelets also contract to pull the edges of the wound close. Most measurements of platelet function involve aggregation. This paper focuses on platelet contraction. Here, we describe a new assay to measure platelets contraction that is repeatable and reproducible. The assay uses standard and common laboratory equipment and can be performed by most laboratory personnel and has the potential to detect clinical pathologies of clot formation. The assay could be developed for bedside patient care where platelet function could be assessed rapidly and assist in the diagnosis of coagulation and platelet disorders.

The regional whole blood program in San Antonio, TX: A 3-year update on prehospital and in-hospital transfusion practices for traumatic and non-traumatic hemorrhage.

Low titer type O Rh-D + whole blood (LTO + WB) has become a first-line resuscitation medium for hemorrhagic shock in many centers around the World. Showing early effectiveness on the battlefield, LTO + WB is used in both the pre-hospital and in-hospital settings for traumatic and non-traumatic hemorrhage resuscitation. Starting in 2018, the San Antonio Whole Blood Collaborative has worked to provide LTO + WB across Southwest Texas, initially in the form of remote damage control resuscitation followed by in-hospital trauma resuscitation. This program has since expanded to include pediatric trauma resuscitation, obstetric hemorrhage, females of childbearing potential, and non-traumatic hemorrhage. The objective of this manuscript is to provide a three-year update on the successes and expansion of this system and outline resuscitation challenges in special populations.

Coagulation function of never frozen liquid plasma stored for 40 days.

BACKGROUND: Never frozen liquid plasma (LP) has limited shelf life versus fresh frozen plasma (FFP) or plasma frozen within 24 h (PF24). Previous studies showed decreasing factor activities after Day (D)14 in thawed FFP but no differences between LP and FFP until D10. This study examined LP function through D40. STUDY DESIGN AND METHODS: FFP and PF24 were stored at -20°C until assaying. LP was assayed on D5 then stored (4°C) for testing through D40. A clinical coagulation analyzer measured Factor (F)V, FVIII, fibrinogen, prothrombin time (PT), and activated partial thromboplastin time (aPTT). Thromboelastography (TEG) and thrombogram measured functional coagulation. Ristocetin cofactor assay quantified von Willebrand factor (vWF) activity. Residual platelets were counted. RESULTS: FV/FVIII showed diminished activity over time in LP, while PT and aPTT both increased over time. LP vWF declined significantly by D7. Fibrinogen remained high through D40. Thrombin lagtime was delayed in LP but consistent to D40, while peak thrombin was significantly lower in LP but did not significantly decline over time. TEG R-time and angle remained constant. LP and PF24 (with residual platelets) had initially higher TEG maximum amplitudes (MA), but by D14 LP was similar to FFP. CONCLUSION: Despite significant declines in some factors in D40 LP, fibrinogen concentration and TEG MA were stable suggesting stored LP provides fibrinogen similarly to frozen plasmas even at D40. LP is easier to store and prepare for prehospital transfusion, important benefits when the alternative is crystalloid.

Cold-stored platelets have better preserved contractile function in comparison with room temperature-stored platelets over 21 days.

Although it is well established that transfusion of platelets in cases of severe bleeding reduces mortality, the availability of platelets is hampered by harsh restrictions on shelf life due to elevated risks of microbial contamination and functional losses with room temperature-stored platelets (RTP) kept at 22°C. In contrast, many recent studies have shown that 4°C cold-stored platelets (CSP) are able to overcome these shortcomings leading to the recent Food and Drug Administration licensure for 14-day stored CSP when conventional platelets are unavailable. This work expands the evidence supporting superiority of CSP function by assaying the less explored platelet-mediated clot retraction of RTP and CSP in either autologous plasma (AP) or platelet additive solution (PAS) for up to 21 days. The results demonstrate that CSP have better preservation of contractile function, exhibiting retraction for up to 21 days in both AP and PAS and forming highly ordered fibrin scaffolds similar to those of fresh platelets. In contrast, RTP stored in AP showed impaired contractile function by Day 5 with no retraction after 10 days, whereas PAS-stored RTP retained contractile function for up to 21 days. Collectively, these findings support extended storage of CSP and suggest that storage in PAS can mitigate functional losses in RTP.

Evaluation of KP-1199: a novel acetaminophen analog for hemostatic function and antinociceptive effects.

BACKGROUND: Acetaminophen (APAP) is a widely self-prescribed analgesic for mild to moderate pain, but overdose or repeat doses can lead to liver injury and death. Kalyra Pharmaceuticals has developed a novel APAP analog, KP-1199, currently in Phase 1 clinical studies, which lacks hepatotoxicity. In this study, the authors evaluated the antinociceptive effect of KP-1199 on thermal injury-induced nociceptive behaviors as well as hemostatic parameters using human blood samples. METHODS: Full-thickness thermal injury was induced in anesthetized adult male Sprague-Dawley rats. On day 7 post-injury, KP-1199 (30 and 60 mg/kg) or APAP (60 mg/kg) was administered orally. Antinociception of KP-1199 and APAP were assessed at multiple time points using Hargreaves' test. In separate experiments, human whole blood was collected and treated with either KP-1199, APAP, or Vehicle (citrate buffer) at 1× (214 µg/ml) and 10× (2140 µg/ml) concentrations. The treated blood samples were assessed for: clotting function, thrombin generation, and platelet activation. RESULTS: APAP did not produce antinociceptive activity. KP-1199 treatment significantly increased the nociceptive threshold, and the antinociceptive activity persisted up to 3 h post-treatment. In human samples, 10× APAP caused significantly prolonged clotting times and increased platelet activation, whereas KP-1199 had caused no negative effects on either parameter tested. CONCLUSION: These results suggest that KP-1199 possesses antinociceptive activity in a rat model of thermal injury. Since KP-1199 does not induce platelet activation or inhibit coagulation, it presents an attractive alternative to APAP for analgesia, especially for battlefield or surgical scenarios where blood loss and blood clotting are of concern.

Cold storage of platelets in platelet additive solution maintains mitochondrial integrity by limiting initiation of apoptosis-mediated pathways.

BACKGROUND: Cold storage of platelets in plasma maintains hemostatic function and is an attractive alternative to room temperature platelets (RTPs). We have recently shown that functional differences between cold-stored platelets (CSPs) and RTPs after 5-day storage are associated with mitochondrial respiration and that CSPs in platelet (PLT) additive solution (PAS) can maintain hemostatic function for at least 15 days. STUDY DESIGN AND METHODS: This study tested the hypothesis that cold storage in PAS preserves mitochondrial integrity by reducing PLT apoptosis. CSPs and RTPs in plasma or PAS were stored and assayed for up to 15 days for mitochondrial function and integrity, mitochondrial-associated mRNA transcript expression, apoptotic proteins, and apoptotic flow cytometry metrics. RESULTS: CSP preserved mitochondria-associated mRNA comparable to baseline levels, improved mitochondrial respiration, and minimized depolarization to Day 15. Additionally, CSPs had minimal induction of caspases, preservation of plasma membrane integrity, and low expression of pro-apoptotic Bax. Storage in PAS appeared to be protective for RTPs in some parameters and enhanced the effects of CSPs. CONCLUSION: Mitochondrial function and molecular analyses defined CSP priming as distinctly different from the well-documented RTP storage lesion. While current blood bank storage at room temperature is limited to 5 to 7 days, refrigeration and storage in PAS for up to 15 days may represent an opportunity to enhance inventories and access to PLT hemostatic support for bleeding patients.

Blood component separation of pathogen-reduced whole blood by the PRP method produces acceptable red cells but platelet yields and function are diminished.

BACKGROUND: This study evaluated blood components processed by the platelet rich plasma (PRP) method from fresh whole blood (FWB) treated with a pathogen reduction technology (PRT). The effects of storage temperature on PRT treated platelet concentrates (PCs) were also examined. STUDY DESIGN AND METHODS: PRT was performed using riboflavin and ultraviolet light on FWB in citrate phosphate dextrose anticoagulant. Following PRT, red blood cells (RBCs), PCs, and plasma for fresh frozen plasma (FFP), were isolated by sequential centrifugation. RBCs were stored at 4°C, FFP at -80°C, and PC at 22°C or at 4°C. Components were assayed throughout their storage times for blood gases, chemistry and CBC, hemostatic function as well as platelet (PLT) and RBC integrity. RESULTS: Component processing following PRT resulted in a significant drop in platelet recovery. Most PRT-PC bags fell below AABB guidelines for platelet count. PRT-PC also showed a decrease in clot strength and decreased aggregometry response. Platelet caspases were activated by PRT. Storage at 4°C improved platelet function. In PRT-FFP, prothrombin time and partial thromboplastin time (PT and aPTT) were prolonged; factors V, VII, VIII, and XI, protein C, and fibrinogen were significantly decreased. Free hemoglobin was elevated two-fold in PRT-RBC. CONCLUSION: Blood components isolated by the PRP method from PRT-treated WB result in a high percentage of PC that fail to meet AABB guidelines. FFP also shows diminished coagulation capacity. However, PRT-RBC are comparable to control-RBC. PRT-WB retains acceptable hemostatic function but alternatives to the PRP method of component separation may be more suitable.

Field-expedient thawing of fresh-frozen plasma.

INTRODUCTION: Frozen plasma is superior to crystalloids for hemorrhage resuscitation but remains logistically challenging in austere environments because of specialized clinical equipment for on-demand thawing. This research examines some ad hoc thawing techniques that have been implemented by military medical personnel. METHODS: Fresh-frozen plasma (FFP) units were thawed accordingly: using a slow cooker (three temperature settings) with preheated or room temperature water; affixing flameless ration heaters from meals ready-to-eat (MREs) to FFP and submerging in water; exposing FFP to electric kettle-boiled water; incubating with a sous vide immersion circulator; or using a clinical thawer (control). Hemostatic function, thrombin generation, factor activities, and essential chemistry were measured after thawing. RESULTS: Even at the highest temperatures, without preheated water the slow cooker doubled thawing time (62.5 min vs. control, 32.5 min; p < 0.0001), and the final temperature was 13.5°C versus 28.8°C in control (p < 0.01). When preheated, the slow cooker thawed in 31.3 minutes (p < 0.05), with a final temperature of 22.4°C. Kettle-boiled water thawed in 23.0 minutes with a final temperature of 25.1°C. The sous vide thawed in 28.1 minutes, with a final temperature of 20.2°C. MRE heaters were insufficient. Functional measures were similar in all conditions. DISCUSSION: In emergencies, protracted plasma thawing is unacceptable, and slower thawing methods also produced cryoprecipitate. Although no functional changes were observed with boiled water thawing, potential negative physiological impacts must be examined. Safe, controlled thawing can be obtained with the sous vide, although optimization requires further testing.

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

BACKGROUND: 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. METHODS: 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. RESULTS: 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. CONCLUSIONS: 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.

Optimizing whole blood storage: hemostatic function of 35-day stored product in CPD, CP2D, and CPDA-1 anticoagulants.

BACKGROUND: Transitioning from whole blood (WB) to components developed from efforts to maximize donor yield. Components are advantageous for specific derangements, but treating hemorrhage with components requires significantly more volume to provide similar effects to WB. Because storage lesion and waste remain problematic, this study examined hemostatic function of refrigerated WB stored for 35 days in anticoagulants citrate-phosphate-dextrose-adenosine (CPDA-1), citrate-phosphate-dextrose (CPD), or citrate-phosphate-double dextrose (CP2D). METHODS: Refrigerated WB units from healthy donors were sampled over 35 days. Global hemostatic parameters were measured by thromboelastometry, thrombogram, platelet aggregometry, and platelet adhesion to collagen under shear conditions. The effects of transfusion filtration and mixing 35-day stored product with fresh WB were evaluated. RESULTS: Countable platelets declined as aggregation clusters appeared in microscopy. While gross platelet agonist-induced aggregation declined over time, normalization revealed aggregation responses in remaining platelets. Peak thrombin generation increased over time. Clot strength diminished over storage in tissue factor-activated samples (normalized by filtration of aggregates). Functional fibrinogen responses remained consistent throughout. Filtration was necessary to maintain consistent platelet adhesion to collagen beyond collection day. Few differences were observed between anticoagulants, and stored/fresh mixing studies normalized coagulation parameters. CONCLUSIONS: WB is easier to collect, store, and transfuse. WB provides platelets, an oft-neglected, critical resuscitation component, but their individual numbers decline as aggregates appear, resulting in diminished coagulation response. WB has better performance in these assays when examined at earlier time points, but expirations designated to specific anticoagulants appear arbitrary for hemostatic functionality, as little changes beyond 21 days regardless of anticoagulant.

Human primary fibroblasts perform similarly to MSCs in assays used to evaluate MSC safety and potency.

BACKGROUND: Cellular therapeutic agents may benefit trauma patients by modulating the immune response to injury, and by reducing inflammation and vascular leakage. Administration of allogeneic mesenchymal stromal cells (MSCs) shows some benefit in preclinical and clinical trials, but less testing has been performed with other cell types. Human primary fibroblasts (FBs) were compared to MSCs in assays designed to evaluate MSCs to determine if these assays actually evaluate properties unique to MSCs or whether related cell types perform similarly. STUDY DESIGN AND METHODS: MSC-related surface marker expression, tissue factor, and human leukocyte antigen-D related were evaluated by flow cytometry, and in vitro adipogenic and osteogenic differentiation potential were determined. Procoagulant activity was determined by thromboelastography. Two potency assays correlated with immunomodulation potential were utilized: the mixed lymphocyte reaction and indoleamine 2,3-dioxygenase enzyme activity assays. RESULTS: Human primary FBs performed similarly to MSCs in assays designed to evaluate MSC characteristics and potency. Although similar for MSC-positive cell surface marker expression, FBs did not show robust adipose differentiation and expressed some level of markers not expected on MSCs. CONCLUSIONS: Human primary FBs are very similar to human MSCs, at least in assays currently used to evaluate MSC potency. Preclinical and clinical testing are required to determine if FBs show similar activity to MSCs in vivo. If FBs show inferior activity in vivo, development of new MSC-specific potency assays will be necessary to evaluate properties relevant to their unique clinical benefits.

Spray-dried plasma deficient in high-molecular-weight multimers of von Willebrand factor retains hemostatic properties.

BACKGROUND: Dried plasmas can overcome logistical barriers that prevent fresh frozen plasma (FFP) usage in acute resuscitation, but processing of these products can detrimentally alter the composition. Spray-dried plasma (SpDP) from single units is deficient in high-molecular-weight multimers of von Willebrand factor (vWF), a critical facilitator of platelet adhesion and thrombus formation. We hypothesized that converting high-molecular-weight multimers to smaller-molecular-weight multimers would retain vWF's capacity to mediate platelet adhesion. STUDY DESIGN AND METHODS: SpDP obtained from untreated FFP was reconstituted with glycine-hydrochloric acid (HCl) and glycine (20 mM:50 mM) or pretreated with glycine-HCl (20 mM) or glycine-glycine-HCl (20 mM:50 mM) and reconstituted with water. In vitro hemostatic potential of SpDPs versus FFP or FFP spiked with 70 mM of glycine was evaluated, leading to a more detailed in vitro study of glycine-HCl-glycine (20 mM:50 mM) pretreated SpDP. Plasmas were combined with RBCs and platelets to observe global coagulation response. RESULTS: While vWF-ristocetin cofactor activity is significantly decreased (-41.13%; p < .0001) in SpDP, a model of vWF-mediated platelet adhesion to collagen under flow showed enhanced function (+13%; p < .01). Fewer microparticles, particularly of platelet origin, were observed in SpDP versus FFP (p < .0001). Small but significant differences in thromboelastography results were observed, although SpDP and FFP were within normal ranges. CONCLUSION: Comparable coagulability was observed in FFP and SpDP. The apparent paradox between vWF-ristocetin cofactor assay and vWF-mediated platelet adhesion may be explained by the increase in smaller multimers of vWF in SpDP, producing different outcomes in these assays.

Evaluation of a lyophilized platelet-derived hemostatic product.

BACKGROUND: Current limitations of platelet shelf life to 5 days have led to an increasingly greater demand for hemostatic agents with greater longevity. The objective of this study was to evaluate the function of a lyophilized platelet-derived hemostatic product (thrombosome [TS]) as a potential alternative to fresh platelets. METHODS: Platelets were collected from whole blood from healthy donors. TSs were reconstituted with water and added to various configurations of reassembled whole blood (platelets, plasma, and RBCs); measures included rotational thromboelastometry (ROTEM), optical aggregometry, mitochondrial function, calibrated automated thrombogram, collagen adhesion under flow (shear flow assay), and flow cytometry. RESULTS: In ROTEM, no differences were observed between maximum clot formation values for contact pathway activation thromboelastometry tests with TSs or platelet samples. Significantly decreased aggregation was observed in the TSs versus platelets (p < 0.001 for all agonists). Flow cytometry measures demonstrated significant decreases in glycoprotein Ib expression and increases in phosphatidylserine expression in the TS group (p < 0.01). The calibrated automated thrombogram assay was suggestive (lag time and peak thrombin) that the TSs might have some thrombogenic properties. Measurements of mitochondrial function revealed that TSs had no functional mitochondria. CONCLUSION: In this study, TSs were shown to have nonfunctional mitochondria. ROTEM measures revealed that the TSs had no impact on clot strength. Likewise, compared to platelets, the TSs displayed minimal aggregation, had significantly more phosphatidylserine (measure of activation status), but had the ability to adhere to a collagen surface under flow conditions and contribute to clot formation and induced greater thrombin generation.

Cold storage of platelets in platelet additive solution: an in vitro comparison of two Food and Drug Administration-approved collection and storage systems.

BACKGROUND: Refrigeration of platelets (PLTs) in a PLT additive solution (PAS) reduces PLT activation compared to storage in plasma and preserves function for at least 15 days. Currently only two PASs are licensed by the Food and Drug Administration, each for use with only one apheresis platform. In this study, we compared the metabolic, functional, and activation status of PLTs collected on a Trima apheresis collection system and stored refrigerated in Isoplate (ISO) PAS to PLTs collected on an Amicus collection system and stored refrigerated in Intersol (INT) PAS. STUDY DESIGN AND METHODS: Apheresis PLTs (n = 4-7 donors) were collected on a Trima in ISO PAS or on an Amicus in INT PAS. PLTs were stored in a walk-in refrigerator (1-6°C) without agitation for long-term storage. Bags were assayed at Days 1, 5, 10, and 15 of storage. Measurements included PLT counts, pH, aggregation response, rotational thromboelastometry, and activation markers. RESULTS: Cold-stored Trima-collected PLTs in ISO were slightly more hemostatic than Amicus-collected PLTs in INT and displayed better adhesion to collagen under flow conditions. Amicus-collected PLTs in INT showed increased microaggregate formation on Days 5 and 10 and a significant decrease in PLT count over storage. Trima-collected PLTs in ISO displayed better clot strength than Amicus-collected PLTs in INT. CONCLUSION: Compared to cold-stored Amicus PLTs in INT, Trima PLTs in ISO display superior in vitro function and may be better suited for treatment of bleeding patients. Clinical studies are warranted to confirm these findings.

Storage of platelets at 4°C in platelet additive solutions prevents aggregate formation and preserves platelet functional responses.

BACKGROUND: Platelet (PLT) storage has been limited to 5 days at room temperature due to metabolic decline and risk for bacterial contamination. Refrigeration preserves PLT metabolism and function as well as limits bacterial growth; however, cold storage of PLTs also leads to aggregate formation. We hypothesized that storage of PLT concentrates at 4°C leads to glycoprotein (GP)IIb-IIIa activation and thus aggregate formation through fibrinogen binding and that this could be prevented by storing PLTs in PLT additive solution (PAS) without compromising PLT function. STUDY DESIGN AND METHODS: Apheresis PLTs in plasma (AP) or apheresis PLTs in PAS were stored at 22 or 4°C for up to 15 days. Measurements include PLT counts, blood gases, aggregation response, flow cytometry analysis of integrin levels, activation markers, and microparticle formation. RESULTS: Storage of AP 4°C led to a gradual decline in PLT count and an increase in aggregate formation that was mediated by intracellular calcium leak and fibrinogen receptor activation. Storage of PAS at 4°C prevented aggregate formation due to dilution of plasma fibrinogen. PAS stored at 4°C maintained aggregation responses to multiple agonists better than 22°C controls. CONCLUSION: Storage of AP at 4°C leads to low level GPIIb-IIIa activation and results in aggregate formation over time. Separating the PLTs from the plasma component and storing them in PAS at 4°C resolves aggregate formation and preserves the metabolic and functional responses of these stored PLTs.