Downscaling platelet cryopreservation: Are platelets frozen in tubes comparable to standard cryopreserved platelets?

BACKGROUND: Platelet cryopreservation extends the shelf-life to at least 2 years. However, platelets are altered during the freeze/thaw process. Downscaling platelet cryopreservation by freezing in tubes would enable rapid screening of novel strategies to improve the quality of cryopreserved platelets (CPPs). The aim of this study was to characterize the effect of freezing conditions on the in vitro phenotype and function of platelets frozen in a low volume compared to standard CPPs. METHODS: Platelets were prepared for cryopreservation using 5%-6% DMSO and processed using standard protocols or aliquoted into 2 mL tubes. Platelets were hyperconcentrated to 25 mL (standard CPPs) or 200 µL (tubes) before freezing at -80°C (n = 8). Six insulators/controlled rate freezing containers were used to vary the freezing rate of platelets in tubes. Platelets were thawed, resuspended in plasma, and then assessed by flow cytometry and thromboelastography. RESULTS: The use of different insulators for tubes changed the freezing rate of platelets compared to platelets frozen using the standard protocol (p < .001). However, this had no impact on the recovery of the platelets (p = .87) or the proportion of platelets expressing GPIbα (p = .46) or GPVI (p = .07), which remained similar between groups. A lower proportion of platelets frozen in tubes externalized phosphatidylserine compared to standard CPPs (p < .001). The clot-forming ability (thromboelastography) of platelets was similar between groups (p > .05). CONCLUSION: Freezing platelets in tubes modified the freezing rate and altered some platelet characteristics. However, the functional characteristics remained comparable, demonstrating the feasibility of downscaling platelet cryopreservation for high-throughput exploratory investigations.

Implementing and Enhancing Social and Economic Needs Screening at a Federally Qualified Health Center.

BACKGROUND: Programs to screen for social and economic needs (SENs) are challenging to implement. AIM: To describe implementation of an SEN screening program for patients obtaining care at a federally qualified health center (FQHC). SETTING: Large Chicago-area FQHC where many patients are Hispanic/Latino and insured through Medicaid. PROGRAM DESCRIPTION: In the program's phase 1 (beginning April 2020), a prescreening question asked about patients' interest in receiving community resources; staff then called interested patients. After several refinements (e.g., increased staffing, tailored reductions in screening frequency) to address challenges such as a large screening backlog, program phase 2 began in February 2021. In phase 2, a second prescreening question asked about patients' preferred modality to learn about community resources (text/email versus phone calls). PROGRAM EVALUATION: During phase 1, 8925 of 29,861 patients (30%) expressed interest in community resources. Only 40% of interested patients were successfully contacted and screened. In phase 2, 5781 of 21,737 patients (27%) expressed interest in resources; 84% of interested patients were successfully contacted by either text/email (43%) or phone (41%). DISCUSSION: Under one-third of patients obtaining care at an FQHC expressed interest in community resources for SENs. After program refinements, rates of follow-up with interested patients substantially increased.

The phenotype of cryopreserved platelets influences the formation of platelet-leukocyte aggregates in an in vitro model.

Cryopreservation significantly alters the phenotype of platelets; generating distinct subpopulations, which may influence the formation of platelet leukocyte aggregates (PLA). PLAs are immunomodulatory and have been associated with transfusion-associated adverse events. As such, the aim of this study was to examine the effect of cryopreservation on the ability of platelets to form PLAs, using a monocyte-like cell line (THP-1). Platelets were tested pre-freeze, post-thaw and following stimulation with TRAP-6 or A23187, both alone and following co-culture with THP-1 cells for 1 and 24 hours (n = 6). Platelet subpopulations and platelet-THP-1 cell aggregates were analyzed using multi-color imaging flow cytometry using Apotracker Green (ApoT), CD42b, CD62P, CD61, and CD45. Cryopreservation resulted in the generation of activated (ApoT-/CD42b+/CD62P+), procoagulant (ApoT+/CD42b+/CD62P+) and a novel (ApoT+/CD42b+/CD62P-) platelet subpopulation. Co-incubation of cryopreserved platelets with THP-1 cells increased PLA formation compared to pre-freeze but not TRAP-6 or A23187 stimulated platelets. P-selectin on the surface membrane was correlated with increased PLA formation. Our findings demonstrate that cryopreservation increases the interaction between platelets and THP-1 cells, largely due to an increase in procoagulant platelets. Further investigation is required to determine the immunological consequences of this interaction.

What do we know? Cryopreserved platelets are an alternative to overcome issues with the short shelf-life of room-temperature stored plateletsAfter thawing, cryopreserved platelets exhibit changes in cell structure and receptor abundanceActivated platelets can attach to leukocytes, forming platelet-leukocyte aggregates and altering their immune functionPlatelet-leukocyte aggregates can increase inflammation, which is associated with adverse events after transfusion, which can negatively affect patient outcomesWhat did we discover? Cryopreservation results in a heterogenous mix of platelet subpopulationsCryopreserved platelets display increased adherence to a monocyte-like cell line (THP-1 cells). Platelet-THP-1 aggregate formation was linked to expression of CD62P on the surface of the plateletsThe increase in cryopreserved platelet-THP-1 cell aggregates was largely due to an increase in procoagulant plateletsWhat is the impact? Our data demonstrate that cryopreservation increases platelet interaction with a monocyte-like cell lineThis may mediate immune responses and/or circulation time of transfused platelets.

The role of sodium citrate during extended cold storage of platelets in platelet additive solutions.

BACKGROUND: Cold-stored platelets are increasingly being used to treat bleeding. Differences in manufacturing processes and storage solutions can affect platelet quality and may influence the shelf life of cold-stored platelets. PAS-E and PAS-F are approved platelet additive solutions (PAS) in Europe and Australia, or the United States respectively. Comparative data are required to facilitate international transferability of laboratory and clinical data. STUDY DESIGN AND METHODS: Single apheresis platelets from matched donors (n = 8) were collected using the Trima apheresis platform and resuspended in either 40% plasma/60% PAS-E or 40% plasma/60% PAS-F. In a secondary study, platelets in PAS-F were supplemented with sodium citrate, to match the concentration in PAS-E. Components were refrigerated (2-6°C) and tested over 21 days. RESULTS: Cold-stored platelets in PAS-F had a lower pH, a greater propensity to form visible (and micro-) aggregates, and higher activation markers compared to PAS-E. These differences were most pronounced during extended storage (14-21 days). While the functional capacity of cold-stored platelets was similar, the PAS-F group displayed minor improvements in ADP-induced aggregation and TEG parameters (R-time, angle). Supplementation of PAS-F with 11 mM sodium citrate improved the platelet content, maintained the pH above specifications and prevented aggregate formation. DISCUSSION: In vitro parameters were similar during short-term cold storage of platelets in PAS-E and PAS-F. Storage in PAS-F beyond 14 days resulted in poorer metabolic and activation parameters. However, the functional capacity was maintained, or even enhanced. The presence of sodium citrate may be an important constituent in PAS for extended cold storage of platelets.

Identification of platelet subpopulations in cryopreserved platelet components using multi-colour imaging flow cytometry.

Cryopreservation of platelets, at - 80 °C with 5-6% DMSO, results in externalisation of phosphatidylserine and the formation of extracellular vesicles (EVs), which may mediate their procoagulant function. The phenotypic features of procoagulant platelets overlap with other platelet subpopulations. The aim of this study was to define the phenotype of in vitro generated platelet subpopulations, and subsequently identify the subpopulations present in cryopreserved components. Fresh platelet components (n = 6 in each group) were either unstimulated as a source of resting platelets; or stimulated with thrombin and collagen to generate a mixture of aggregatory and procoagulant platelets; calcium ionophore (A23187) to generate procoagulant platelets; or ABT-737 to generate apoptotic platelets. Platelet components (n = 6) were cryopreserved with DMSO, thawed and resuspended in a unit of thawed plasma. Multi-colour panels of fluorescent antibodies and dyes were used to identify the features of subpopulations by imaging flow cytometry. A combination of annexin-V (AnnV), CD42b, and either PAC1 or CD62P was able to distinguish the four subpopulations. Cryopreserved platelets contained procoagulant platelets (AnnV+/PAC1-/CD42b+/CD62P+) and a novel population (AnnV+/PAC1-/CD42b+/CD62P-) that did not align with the phenotype of aggregatory (AnnV-/PAC1+/CD42b+/CD62P+) or apoptotic (AnnV+/PAC1-/CD42b-/CD62P-) subpopulations. These data suggests that the enhanced haemostatic potential of cryopreserved platelets may be due to the cryo-induced development of procoagulant platelets, and that additional subpopulations may exist.

Assessment of the soluble proteins HMGB1, CD40L and CD62P during various platelet preparation processes and the storage of platelet concentrates: The BEST collaborative study.

BACKGROUND: Structural and biochemical changes in stored platelets are influenced by collection and processing methods. This international study investigates the effects of platelet (PLT) processing and storage conditions on HMGB1, sCD40L, and sCD62P protein levels in platelet concentrate supernatants (PCs). STUDY DESIGN/METHODS: PC supernatants (n = 3748) were collected by each international centre using identical centrifugation methods (n = 9) and tested centrally using the ELISA/Luminex platform. Apheresis versus the buffy coat (BC-PC) method, plasma storage versus PAS and RT storage versus cold (4°C) were investigated. We focused on PC preparation collecting samples during early (RT: day 1-3; cold: day 1-5) and late (RT: day 4-7; cold: day 7-10) storage time points. RESULTS: HMGB1, sCD40L, and sCD62P concentrations were similar during early storage periods, regardless of storage solution (BC-PC plasma and BC-PC PAS-E) or temperature. During storage and without PAS, sCD40L and CD62P in BC-PC supernatants increased significantly (+33% and +41%, respectively) depending on storage temperature (22 vs. 4°C). However, without PAS-E, levels decreased significantly (-31% and -20%, respectively), depending on storage temperature (22 vs. 4°C). Contrastingly, the processing method appeared to have greater impact on HMGB1 release versus storage duration. These data highlight increases in these parameters during storage and differences between preparation methods and storage temperatures. CONCLUSIONS: The HMGB1 release mechanism/intracellular pathways appear to differ from sCD62P and sCD40L. The extent to which these differences affect patient outcomes, particularly post-transfusion platelet increment and adverse events, warrants further investigation in clinical trials with various therapeutic indications.

Gamma and X-ray irradiation do not affect the in vitro quality of refrigerated apheresis platelets in platelet additive solution (PAS-E).

BACKGROUND: Platelet refrigeration (cold storage) provides the advantages of an extended shelf life and reduces the risk of bacterial growth, compared to platelets stored at room temperature (RT). However, processing modifications, such as irradiation, may further improve the safety and/or alter the quality of cold-stored platelets. Platelet components are irradiated to prevent transfusion-associated graft versus host disease (TA-GvHD) in high-risk patients; and while irradiation has little effect on the quality of RT-stored platelet components, there is no data assessing the effect irradiation has following cold storage. STUDY DESIGN AND METHODS: Triple-dose apheresis platelets were collected in 40% plasma/60% PAS-E, using the TRIMA apheresis platform, and refrigerated (2-6°C) within 8 h of collection. On day 2, one of each component was gamma or X-ray irradiated or remained non-irradiated. Platelets were tested over 21 days. RESULTS: The platelet concentration decreased by approximately 20% in all groups during 21 days of storage (p > .05). Irradiation (gamma or X-ray) did not affect platelet metabolism, and the pH was maintained above the minimum specification (>6.4) for 21 days. The surface phenotype and the composition of the supernatant was similar in non-irradiated and irradiated platelets, regardless of the source of radiation. Functional responses (aggregation and clot formation) were not affected by irradiation. DISCUSSION: Gamma and X-ray irradiation do not affect the in vitro quality of platelet components stored in the cold for up to 21 days. This demonstrates the acceptability of irradiating cold-stored platelets, which has the potential to improve their safety for at-risk patient cohorts.

Cold storage alters the immune characteristics of platelets and potentiates bacterial-induced aggregation.

BACKGROUND AND OBJECTIVES: Cold-stored platelets are currently under clinical evaluation and have been approved for limited clinical use in the United States. Most studies have focused on the haemostatic functionality of cold-stored platelets; however, limited information is available examining changes to their immune function. MATERIALS AND METHODS: Two buffy-coat-derived platelet components were combined and split into two treatment arms: room temperature (RT)-stored (20-24°C) or refrigerated (cold-stored, 2-6°C). The concentration of select soluble factors was measured in the supernatant using commercial ELISA kits. The abundance of surface receptors associated with immunological function was assessed by flow cytometry. Platelet aggregation was assessed in response to Escherichia coli and Staphylococcus aureus, in the presence and absence of RGDS (blocks active conformation of integrin α2 ß3 ). RESULTS: Cold-stored platelet components contained a lower supernatant concentration of C3a, RANTES and PF4. The abundance of surface-bound P-selectin and integrin α2 ß3 in the activated conformation increased during cold storage. In comparison, the abundance of CD86, CD44, ICAM-2, CD40, TLR1, TLR2, TLR4, TLR3, TLR7 and TLR9 was lower on the surface membrane of cold-stored platelets compared to RT-stored components. Cold-stored platelets exhibited an increased responsiveness to E. coli- and S. aureus-induced aggregation compared to RT-stored platelets. Inhibition of the active conformation of integrin α2 ß3 using RGDS reduced the potentiation of bacterial-induced aggregation in cold-stored platelets. CONCLUSION: Our data highlight that cold storage changes the in vitro immune characteristics of platelets, including their sensitivity to bacterial-induced aggregation. Changes in these immune characteristics may have clinical implications post transfusion.

Cryopreserved platelets compared with liquid-stored platelets for the treatment of surgical bleeding: protocol for two multicentre randomised controlled blinded non-inferiority trials (the CLIP-II and CLIPNZ-II trials).

INTRODUCTION: Cryopreservation at -80°C in dimethylsulphoxide extends platelet shelf-life from 7 days to 2 years. Only limited comparative trial data supports the safety and effectiveness of cryopreserved platelets as a treatment for surgical bleeding. Cryopreserved platelets are not currently registered for civilian use in most countries. METHODS AND ANALYSIS: CLIP-II and CLIPNZ-II are harmonised, blinded, multicentre, randomised, controlled clinical non-inferiority trials comparing bleeding, transfusion, safety and cost outcomes associated with cryopreserved platelets versus conventional liquid platelets as treatment for bleeding in cardiac surgery. CLIP-II is planning to enrol patients in 12 tertiary hospitals in Australia; CLIPNZ-II will recruit in five tertiary hospitals in New Zealand. The trials use near-identical protocols aside from details of cryopreserved platelet preparation. Patients identified preoperatively as being at high risk of requiring a platelet transfusion receive up to three units of study platelets if their treating doctor considers platelet transfusion is indicated. The primary endpoint is blood loss through the surgical drains in the 24 hours following intensive care unit (ICU) admission after surgery. Other endpoints are blood loss at other time points, potential complications, adverse reactions, transfusion and fluid requirement, requirement for procoagulant treatments, time to commencement of postoperative anticoagulants, delay between platelet order and commencement of infusion, need for reoperation, laboratory and point-of-care clotting indices, cost, length of mechanical ventilation, ICU and hospital stay, and mortality. Transfusing 202 (CLIP-II) or 228 (CLIPNZ-II) patients with study platelets will provide 90% power to exclude the possibility of greater than 20% inferiority in the primary endpoint. If cryopreserved platelets are not inferior to liquid-stored platelets, the advantages of longer shelf-life would justify rapid change in clinical practice. Cost-effectiveness analyses will be incorporated into each study such that, should clinical non-inferiority compared with standard care be demonstrated, the hospitals in each country that would benefit most from changing to a cryopreserved platelet blood bank will be known. ETHICS AND DISSEMINATION: CLIP-II was approved by the Austin Health Human Research Ethics Committee (HREC/54406/Austin-2019) and by the Australian Red Cross Lifeblood Ethics Committee (2019#23). CLIPNZ-II was approved by the New Zealand Southern Health and Disability Ethics Committee (21/STH/66). Eligible patients are approached for informed consent at least 1 day prior to surgery. There is no provision for consent provided by a substitute decision-maker. The results of the two trials will be submitted separately for publication in peer-reviewed journals. TRIAL REGISTRATION NUMBERS: NCT03991481 and ACTRN12621000271808.

A pilot randomized clinical trial of cryopreserved versus liquid-stored platelet transfusion for bleeding in cardiac surgery: The cryopreserved versus liquid platelet-New Zealand pilot trial.

BACKGROUND AND OBJECTIVES: Platelets for transfusion have a shelf-life of 7 days, limiting availability and leading to wastage. Cryopreservation at -80°C extends shelf-life to at least 1 year, but safety and effectiveness are uncertain. MATERIALS AND METHODS: This single centre blinded pilot trial enrolled adult cardiac surgery patients who were at high risk of platelet transfusion. If treating clinicians determined platelet transfusion was required, up to three units of either cryopreserved or liquid-stored platelets intraoperatively or during intensive care unit admission were administered. The primary outcome was protocol safety and feasibility. RESULTS: Over 13 months, 89 patients were randomized, 23 (25.8%) of whom received a platelet transfusion. There were no differences in median blood loss up to 48 h between study groups, or in the quantities of study platelets or other blood components transfused. The median platelet concentration on the day after surgery was lower in the cryopreserved platelet group (122 × 103 /µl vs. 157 × 103 /µl, median difference 39.5 ×103 /µl, p = 0.03). There were no differences in any of the recorded safety outcomes, and no adverse events were reported on any patient. Multivariable adjustment for imbalances in baseline patient characteristics did not find study group to be a predictor of 24-h blood loss, red cell transfusion or a composite bleeding outcome. CONCLUSION: This pilot randomized controlled trial demonstrated the feasibility of the protocol and adds to accumulating data supporting the safety of this intervention. Given the clear advantage of prolonged shelf-life, particularly for regional hospitals in New Zealand, a definitive non-inferiority phase III trial is warranted.

Cryopreservation alters the immune characteristics of platelets.

BACKGROUND: Cryopreserved platelets are under clinical evaluation as they offer improvements in shelf-life and potentially hemostatic effectiveness. However, the effect of cryopreservation on characteristics related to the immune function of platelets has not been examined. STUDY DESIGN AND METHODS: Buffy coat derived platelets were cryopreserved at -80°C using 5%-6% dimethylsulfoxide (DMSO, n = 8). Paired testing was conducted pre-freeze (PF), post-thaw (PT0), and after 24 h of post-thaw storage at room temperature (PT24). The concentration of biological response modifiers (BRMs) in the supernatant was measured using commercial ELISAs and surface receptor abundance was assessed by flow cytometry. RESULTS: Cryopreservation resulted in increased RANTES, PF4, and C3a but decreased IL-1ß, OX40L, IL-13, IL-27, CD40L, and C5a concentrations in the supernatant, compared to PF samples. C4a, endocan, and HMGB1 concentrations were similar between the PF and PT0 groups. The abundance of surface-expressed P-selectin, siglec-7, TLR3, TLR7, and TLR9 was increased PT0; while CD40, CLEC2, ICAM-2, and MHC-I were decreased, compared to PF. The surface abundance of CD40L, B7-2, DC-SIGN, HCAM, TLR1, TLR2, TLR4, and TLR6 was unchanged by cryopreservation. Following 24 h of post-thaw storage, all immune associated receptors and TLRs increased to levels higher than observed on PF and PT0 platelets. CONCLUSION: Cryopreservation alters the immune phenotype of platelets. Understanding the clinical implications of the observed changes in BRM release and receptor abundance are essential, as they may influence the likelihood of adverse events.

Platelet procoagulant potential is reduced in platelet concentrates ex vivo but appears restored following transfusion.

BACKGROUND: The procoagulant profile of platelet concentrates (PCs) following transfusion has been difficult to evaluate due to lack of specific markers. This study aimed to characterize procoagulant platelets in PCs and the effect of transfusion. STUDY DESIGN AND METHODS: Buffy coat-derived PCs from 12 donors were pooled, split, then stored conventionally, cold (2-6°C) or cryopreserved (-80°C). Procoagulant platelet profiles were assessed by flow cytometry (GSAO+ /P-selectin+ ), lactadherin-binding, and calibrated automated thrombogram, during storage, unstimulated, or after thrombin and collagen stimulation and compared with blood from healthy volunteers. Platelet activation (P-selectin) and procoagulant platelet formation potential were measured (flow cytometry) in patients receiving clinically indicated conventional PC transfusion. RESULTS: Independent of significant increases with storage, procoagulant platelet proportions with and without agonist stimulation were significantly blunted in conventionally stored PCs (stimulated day 5 conventional PC 4.2 ± 1.3%, healthy volunteer blood 11.1 ± 2.9%; p < .0001). Cryopreserved PCs contained the highest proportion of procoagulant platelets (unstimulated: cryopreserved 25.6 ± 1.8% vs. day 5 conventional 0.5 ± 0.1% vs. day 14 cold-stored 5.8 ± 1.0%, p < .0001), but demonstrated minimal increase with agonist. Transfusion of PCs was associated with an increase in procoagulant platelets (2.2 ± 1.4% vs. 0.6 ± 0.2%; p = .004) and reversal of the blunted agonist response (15.8 ± 5.9% vs. 4.0 ± 1.6%; p < .0001). Procoagulant responses post-transfusion were significantly higher than healthy controls, suggesting a priming effect. The P-selectin agonist response was not restored upon transfusion (79.4 ± 13.9% vs. 82.0 ± 2.5%). CONCLUSION: Storage blunts the procoagulant platelet response to agonist stimulation in PCs. Despite this, conventionally stored PCs have high procoagulant potential following transfusion, with a discordant, persistent reduction in P-selectin response.

The in vitro quality of X-irradiated platelet components in PAS-E is equivalent to gamma-irradiated components.

BACKGROUND: Blood components are irradiated to inactivate lymphocytes in an effort to prevent transfusion-associated graft versus host disease. Although gamma irradiators are commonly used, they are subjected to rigorous health, safety, and compliance regulations, compared with X-irradiators which have the advantage of only emitting radiation while the machine is switched on. While the effects of gamma irradiation on platelet components are well known, there is little or no data comparing the effects of X- and gamma-irradiation on the quality of these components. Therefore, this study examined the in vitro quality of platelet components (pooled and apheresis) following X- or gamma-irradiation. STUDY DESIGN AND METHODS: Whole-blood-derived (pooled) and apheresis platelet components in platelet additive solution (n = 20 pairs for each type) were irradiated (X vs. gamma). In vitro platelet quality was tested prior to irradiation (day 1) and subsequently on days 2, 5, and 7. Non-irradiated components were tested on day 5 in parallel as reference controls. Metabolic parameters, surface expression of glycoproteins and activation markers (CD62P and annexin-V binding), and agonist-induced aggregation were measured. RESULTS: All components met Council of Europe specifications. There were no statistical differences in any in vitro quality measurements between X- and gamma-irradiated pooled or apheresis platelet components. CONCLUSION: X- and gamma-irradiation have similar effects on the in vitro quality of stored blood components, indicating that either technology represents a suitable option for irradiation of platelet components.

Refrigeration of apheresis platelets in platelet additive solution (PAS-E) supports in vitro platelet quality to maximize the shelf-life.

BACKGROUND: Refrigeration, or cold-storage, of platelets may be beneficial to extend the limited shelf-life of conventionally stored platelets and support transfusion protocols in rural and military areas. The aim of this study was to compare the morphologic, metabolic, and functional aspects of apheresis platelets stored at room-temperature (RT) or cold conditions, in either plasma or supplemented with platelet additive solution (PAS). STUDY DESIGN AND METHODS: Double-dose apheresis platelets were collected in either 100% plasma or 40% plasma/60% PAS-E using the Trima apheresis platform. One component from each group was either stored at RT (20-24°C) or refrigerated (2-6°C). Platelets were tested over a 21-day period. RESULTS: The platelet concentration decreased by approximately 30% in all groups during 21 days of storage (p > .05). Cold-storage reduced glycolytic metabolism, and the pH was maintained above the minimum specification (>6.4) for 21 days only when platelets were stored in PAS. The surface phenotype and the composition of the supernatant were differentially affected by temperature and storage solution. Functional responses (aggregation, agonist-induced receptor activation, clotting time) were improved during cold-storage, and the influence of residual plasma was assay dependent. CONCLUSION: In vitro platelet quality is differentially affected by storage time, temperature, and solution. Cold-storage, particularly in PAS, better maintains key metabolic, phenotypic, and functional parameters during prolonged storage.

Reconstituted cryopreserved platelets synthesize proteins during short-term storage and packaging a defined subset into microvesicles.

BACKGROUND: Cryopreservation of platelets (PLTs) could allow extension of their shelf-life to years, compared to days for liquid stored platelets. Due to their greater hemostatic effect, reconstituted cryopreserved platelets (cryo-PLTs) would be able to support bleeding emergencies. Since protein synthesis has been linked to PLT functions, such as clot formation and immune responses, the translational capacity of reconstituted cryo-PLTs was assessed upon thawing and short-term storage. METHODS/MATERIALS: Platelets were frozen at -80°C with 5-6% DMSO. Upon thawing, they were reconstituted in plasma and then aliquoted (12 ml) into mini-bags and assessed over 24 h of storage at RT. One series served as control; the second and third series were spiked with either 300 µM puromycin (Pm) or 227 nM biotin-labeled Pm. Samples were tested for in vitro quality and PLT microvesicle enumeration by flow cytometry. Protein synthesis in cryo-PLTs was assessed using a modified method based on puromycin-associated nascent chain proteomics. RESULTS: In vitro parameters of reconstituted and subsequently stored platelets were consistent with previously published results. Mass-spectrometry analyses identified that 22 proteins were synthesized in PLTs and 13 of those were observed in platelet microvesicles (PMVs). CONCLUSION: Cryo-PLTs can synthesize proteins upon reconstitution and storage. Discovery of a subset of these proteins in the PMV suggests a role in vesicle encapsulation, possibly in a selective manner. This observation provides novel insights into the capacity for protein synthesis in cryo-PLTs and the potential regulation of protein packaging into PMV.

Effect of Recombinant Human Perlecan Domain V Tethering Method on Protein Orientation and Blood Contacting Activity on Polyvinyl Chloride.

Surface modification of biomaterials is a promising approach to control biofunctionality while retaining the bulk biomaterial properties. Perlecan is the major proteoglycan in the vascular basement membrane that supports low levels of platelet adhesion but not activation. Thus, perlecan is a promising bioactive for blood-contacting applications. This study furthers the mechanistic understanding of platelet interactions with perlecan by establishing that platelets utilize domains III and V of the core protein for adhesion. Polyvinyl chloride (PVC) is functionalized with recombinant human perlecan domain V (rDV) to explore the effect of the tethering method on proteoglycan orientation and bioactivity. Tethering of rDV to PVC is achieved via either physisorption or covalent attachment via plasma immersion ion implantation (PIII) treatment. Both methods of rDV tethering reduce platelet adhesion and activation compared to the pristine PVC, however, the mechanisms are unique for each tethering method. Physisorption of rDV on PVC orientates the molecule to hinder access to the integrin-binding region, which inhibits platelet adhesion. In contrast, PIII treatment orientates rDV to allow access to the integrin-binding region, which is rendered antiadhesive to platelets via the glycosaminoglycan (GAG) chain. These effects demonstrate the potential of rDV biofunctionalization to modulate platelet interactions for blood contacting applications.

The immune potential of ex vivo stored platelets: a review.

Platelets are now acknowledged as key regulators of the immune system, as they are capable of mediating inflammation, leucocyte recruitment and activation. This activity is facilitated through platelet activation, which induces significant changes in the surface receptor profile and triggers the release of a range of soluble biological response modifiers (BRMs). In the field of transfusion medicine, the immune function of platelets has gained considerable attention as this may be linked to the development of adverse transfusion reactions. Further, component manufacturing and storage methodologies may impact the immunoregulatory role of platelets, and an understanding of this impact is crucial and should be considered alongside their haemostatic characteristics. This review highlights the key interactions between platelets and traditional immune modulators. Further, the potential impact of current and novel component storage methodologies, such as refrigeration and cryopreservation, on this functional capacity is examined, highlighting why further knowledge in this area would be of benefit.

Extended storage of thawed platelets: Refrigeration supports postthaw quality for 10 days.

BACKGROUND: Cryopreservation of platelets with dimethylsulfoxide (DMSO) at -80°C increases their shelf life from days to years. Once thawed, platelets are stored at room temperature (RT), and the shelf life is limited to 4-6 hours. However, refrigeration (cold storage) may facilitate a prolongation of the shelf life of thawed platelets. STUDY DESIGN AND METHODS: ABO-matched buffy coat-derived platelets (30% plasma/70% SSP+) were cryopreserved at -80°C in 5%-6% DMSO. Paired cryopreserved platelet components were thawed, resuspended in 30% plasma/70% SSP+, and then stored at either 20°C-24°C with agitation (RT) or at 2°C-6°C (cold). In vitro platelet quality was assessed over 10 days of postthaw storage. RESULTS: During postthaw storage, the platelet concentration of RT-stored components decreased significantly more than components in cold storage (Day 10 RT 58 ± 10 × 109 /unit vs Day 10 cold 142 ± 16 × 109 /unit; P < .0001). Cold storage reduced the metabolic rate of thawed platelets. During storage, the surface glycoprotein ([GP] Ibα, GPVI, GPIIb, GPIIIa) and activation marker (P-selectin and phosphatidylserine) profile of cold platelets was closer to freshly thawed platelets (Day 0) than those stored at RT. Thromboelastography (reaction time) demonstrated that the procoagulant nature of cryopreserved platelets was preserved during 10 days of cold storage, while RT-stored thawed platelets displayed a gradual prolongation of the time taken to initiate clot formation. CONCLUSION: Cold storage of thawed platelets preserves the platelet phenotype and function for up to 10 days, compared to thawed platelets stored at RT. Thus, cold storage of thawed platelets may represent a simple approach to extend the postthaw shelf life.

Characterizing the ability of an ice recrystallization inhibitor to improve platelet cryopreservation.

Improving aspects of platelet cryopreservation would help ease logistical challenges and potentially expand the utility of frozen platelets. Current cryopreservation procedures damage platelets, which may be caused by ice recrystallization. We hypothesized that the addition of a small molecule ice recrystallization inhibitor (IRI) to platelets prior to freezing may reduce cryopreservation-induced damage and/or improve the logistics of freezing and storage. Platelets were frozen using standard conditions of 5-6% dimethyl sulfoxide (Me2SO) or with supplementation of an IRI, N-(2-fluorophenyl)-d-gluconamide (2FA), prior to storage at -80 °C. Alternatively, platelets were frozen with 5-6% Me2SO at -30 °C or with 3% Me2SO at -80 °C with or without 2FA supplementation. Supplementation of platelets with 2FA improved platelet recovery following storage under standard conditions (p = 0.0017) and with 3% Me2SO (p = 0.0461) but not at -30 °C (p = 0.0835). 2FA supplementation was protective for GPVI expression under standard conditions (p = 0.0011) and with 3% Me2SO (p = 0.0042). Markers of platelet activation, such as phosphatidylserine externalization and microparticle release, were increased following storage at -30 °C or with 3% Me2SO, and 2FA showed no protective effect. Platelet function remained similar regardless of 2FA, although functionality was reduced following storage at -30 °C or with 3% Me2SO compared to standard cryopreserved platelets. While the addition of 2FA to platelets provided a small level of protection for some quality parameters, it was unable to prevent alterations to the majority of in vitro parameters. Therefore, it is unlikely that ice recrystallization is the major cause of cryopreservation-induced damage.

Calcium chelation: a novel approach to reduce cryopreservation-induced damage to frozen platelets.

BACKGROUND: Cryopreserved platelets are phenotypically and functionally different to conventionally stored platelets. Calcium may be released from internal stores during the freeze-thaw process, initiating signaling events which lead to these alterations. It was hypothesized that the addition of a calcium chelator prior to cryopreservation may mitigate some of these changes. METHODS: Buffy coat-derived platelets that had been pooled and split were tested fresh and following cryopreservation (n = 8 per group). Platelets were cryopreserved using 5%-6% dimethylsulfoxide (DMSO) or were supplemented with increasing concentrations of the internal calcium chelator, BAPTA-AM (100 µM, 200 µM, or 400 µM), prior to storage at -80°C. RESULTS: Supplementation of platelets with BAPTA-AM prior to freezing improved platelet recovery in a dose response manner (400 µM: 84 ± 2%) compared to standard DMSO cryopreserved platelets (70 ± 4%). There was a loss of GPIbα, GPVI, and GPIIb/IIIa receptors on platelets following cryopreservation, which was rescued when platelets were supplemented with BAPTA-AM (400 µM: p < 0.0001 for all). Platelet activation markers, such as phosphatidylserine and P-selectin, were externalized on platelets following cryopreservation. However, the addition of BAPTA-AM significantly reduced the increase of these activation markers on cryopreserved platelets (400 µM: p < 0.0001 for both). Both cryopreserved platelet groups exhibited similar functionality as assessed by thromboelastography, forming clots at a faster rate than fresh platelets. CONCLUSIONS: This study demonstrates that calcium plays a crucial role in mediating cryopreservation-induced damage to frozen platelets. The addition of the calcium chelator, BAPTA-AM, prior to cryopreservation reduces this damage.