Cryopreservation of mesenchymal stem/stromal cells using a DMSO-free solution is comparable to DMSO-containing cryoprotectants: results of an international multicenter PACT/BEST collaborative study.

BACKGROUND AND AIM: An essential aspect of ensuring availability and stability of mesenchymal stem/stromal cells (MSCs) products for clinical use is that these cells are cryopreserved before individual infusion into patients. Currently, cryopreservation of MSCs involves use of a cryoprotectant solution containing dimethyl sulfoxide (DMSO). However, it is recognized that DMSO may be toxic for both the patient and the MSC product. In this Production Assistance for Cellular Therapies (PACT) and Biomedical Excellence for Safer Transfusion (BEST) Collaborative study, we compared a novel DMSO-free solution with DMSO containing cryoprotectant solutions for freezing MSCs. METHODS: A DMSO-free cryoprotectant solution containing sucrose, glycerol, and isoleucine (SGI) in a base of Plasmalyte A was prepared at the University of Minnesota. Cryoprotectant solutions containing 5-10% DMSO (in-house) were prepared at seven participating centers (five from USA, one each from Australia and Germany). The MSCs were isolated from bone marrow or adipose tissue and cultured ex vivo per local protocols at each center. The cells in suspension were frozen by aliquoting into vials/bags. For six out of the seven centers, the vials/bags were placed in a controlled rate freezer (one center placed them at -80°C freezer overnight) before transferring to liquid nitrogen. The cells were kept frozen for at least one week before thawing and testing. Pre- and post-thaw assessment included cell viability and recovery, immunophenotype as well as transcriptional and gene expression profiles. Linear regression, mixed effects models and two-sided t-tests were applied for statistical analysis. RESULTS: MSCs had an average viability of 94.3% (95% CI: 87.2-100%) before cryopreservation, decreasing by 4.5% (95% CI: 0.03-9.0%; P: 0.049) and 11.4% (95% CI: 6.9-15.8%; P< 0.001), for MSCs cryopreserved in the in-house and SGI solutions, respectively. The average recovery of viable MSCs cryopreserved in the SGI was 92.9% (95% CI: 85.7-100.0%), and it was lower by 5.6% (95% CI: 1.3-9.8%, P < 0.013) for the in-house solution. Additionally, MSCs cryopreserved in the two solutions had expected level of expressions for CD45, CD73, CD90, and CD105 with no significant difference in global gene expression profiles. CONCLUSION: MSCs cryopreserved in a DMSO-free solution containing sucrose, glycerol, and isoleucine in a base of Plasmalyte A had slightly lower cell viability, better recovery, and comparable immunophenotype and global gene expression profiles compared to MSCs cryopreserved in DMSO containing solutions. The average viability of MSCs in the novel solution was above 80% and, thus, likely clinically acceptable. Future studies are suggested to test the post-thaw functions of MSCs cryopreserved in the novel DMSO-free solution.

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.

A review and analysis of outcomes in randomized clinical trials of plasma transfusion in patients with bleeding or for the prevention of bleeding: The BEST collaborative study.

BACKGROUND: Previous systematic reviews have revealed an inconsistency of outcome definitions as a major barrier in providing evidence-based guidance for the use of plasma transfusion to prevent or treat bleeding. We reviewed and analyzed outcomes in randomized controlled trials (RCTs) to provide a methodology for describing and classifying outcomes. STUDY DESIGN AND METHODS: RCTs involving transfusion of plasma published after 2000 were identified from a prior review (Yang 2012) and combined with an updated systematic literature search of multiple databases (July 1, 2011 to January 17, 2023). Inclusion of publications, data extraction, and risk of bias assessments were performed in duplicate. (PROSPERO registration number is: CRD42020158581). RESULTS: In total, 5579 citations were identified in the new systematic search and 22 were included. Six additional trials were identified from the previous review, resulting in a total of 28 trials: 23 therapeutic and five prophylactic studies. An increasing number of studies in the setting of major bleeding such as in cardiovascular surgery and trauma were identified. Eighty-seven outcomes were reported with a mean of 11 (min-max. 4-32) per study. There was substantial variation in outcomes used with a preponderance of surrogate measures for clinical effect such as laboratory parameters and blood usage. CONCLUSION: There is an expanding literature on plasma transfusion to inform guidelines. However, considerable heterogeneity of reported outcomes constrains comparisons. A core outcome set should be developed for plasma transfusion studies. Standardization of outcomes will motivate better study design, facilitate comparison, and improve clinical relevance for future trials of plasma transfusion.

Extending the post-thaw shelf-life of cryoprecipitate when stored at refrigerated temperatures.

BACKGROUND AND OBJECTIVES: The post-thaw shelf-life of cryoprecipitate is 6 h, leading to high wastage. Storage of thawed cryoprecipitate at refrigerated temperatures may be feasible to extend the shelf-life. This study aimed to evaluate the quality of thawed cryoprecipitate stored at 1-6°C for up to 14 days. MATERIALS AND METHODS: Cryoprecipitate (mini- and full-size packs derived from both apheresis and whole blood [WB] collections) was thawed, immediately sampled and then stored at 1-6°C for up to 14 days. Mini-packs were sampled at 6, 24, 48 and 72 h, day 7 and 14; full-size cryoprecipitate was sampled on day 3, 5 or 7. Coagulation factors (F) II, V, VIII, IX, X and XIII, von Willebrand factor (VWF) and fibrinogen were measured using a coagulation analyser. Thrombin generation was measured by calibrated automated thrombogram. RESULTS: FVIII decreased during post-thaw storage; this was significant after 24 h for WB (p = 0.0002) and apheresis (p < 0.0001). All apheresis and eight of 20 WB cryoprecipitate met the FVIII specification (≥ 70 IU/unit) on day 14 post-thaw. Fibrinogen remained stable for 48 h, and components met the specification on day 14 post-thaw. There were no significant differences in VWF (WB p = 0.1292; apheresis p = 0.1507) throughout storage. There were small but significant decreases in thrombin generation lag time, endogenous thrombin potential and time to peak for both WB and apheresis cryoprecipitate. CONCLUSION: Whilst coagulation factors in cryoprecipitate decreased after post-thaw storage, the thawed cryoprecipitate met the Council of Europe specifications when stored at refrigerated temperatures for 7 days.

Platelets retain function and can be stored following disruption of human leucocyte antigens.

BACKGROUND AND OBJECTIVES: Antibodies to human leucocyte antigen (HLA) Class-I antigens can lead to refractoriness to platelet transfusion. Although this can be overcome by transfusion of HLA-compatible platelets, they are not always available. Disruption of HLA antigens on platelets by acid treatment may be a suitable alternative when no other components are available. The aim of this study was to assess the effect of HLA disruption and subsequent storage of platelet components. MATERIALS AND METHODS: Platelet components were treated with 0.9% saline or citric acid solution (pH 3.0), and then stored until expiry (Day 7). HLA and platelet glycoprotein expression, platelet viability, activation and sialylation were measured by flow cytometry. Release of soluble factors was measured by ELISA and metabolism by biochemistry analyser. Reactivity to patient anti-sera containing anti-HLA antibodies was measured using platelet immunofluorescence tests (PIFTs) and monoclonal antibody immobilization of platelet antigen (MAIPA) assays. Platelet function was measured using aggregometry and thromboelastography (TEG). RESULTS: Acid treatment reduced detection of HLA Class-I on platelets by 75%, with significant reductions in reactivity to patient anti-sera. Acid treatment reduced platelet content and viability, increased platelet activation and accelerated metabolism. Glycan cleavage was increased by acid treatment. Treatment reduced platelet activation following agonist stimulation by ADP and TRAP-6, but platelets remained functional, displaying increased aggregation response and reduced time to clot formation by TEG. CONCLUSION: Although HLA disruption had some detrimental effects, acid-treated platelets remained functional, retaining their capacity to respond to agonists and form clots, and with further development could be used to support refractory patients.

Haemolysis in red blood cell components is associated with donor ferritin and body mass index status, but not donation frequency.

Whole blood donors who donate more frequency are more likely to develop iron deficiency, which could potentially affect the quality of the red blood cell (RBC) components during storage. Additional donor factors such as sex, age at donation, donor body mass index (BMI), as well as the manufacturing method could also affect RBC component quality, particularly haemolysis. The aim of this study was to examine the relationship between donation frequency, donor ferritin levels and BMI status on an extensive set of RBC characteristics in vitro, during storage at 2-6 °C for 42 days. A whole blood donation was collected from 787 Australian blood donors, held overnight, before top-and-bottom separation to produce RBC components. RBC components were tested using a panel of in vitro assays. Serum ferritin was tested from a sample taken at the time of donation, and donor demographic data was collected. Haemolysis in RBC components was not found to be associated with donation frequency. Increased red cell haemolysis, lactate concentration, extracellular potassium and RBC-derived microparticle numbers were significantly associated with a high BMI in male donors. There was also a trend towards increased red cell haemolysis in donors with ferritin concentrations in the upper range. Our findings indicate that although older male donors with potentially higher BMI are able to donate whole blood quite frequently, the resultant RBC components may have poorer in vitro quality.

Lipidomic changes occurring in platelets during extended cold storage.

OBJECTIVES: Cold storage is being implemented as an alternative to conventional room-temperature storage for extending the shelf-life of platelet components beyond 5-7 days. The aim of this study was to characterise the lipid profile of platelets stored under standard room-temperature or cold (refrigerated) conditions. METHODS: Matched apheresis derived platelet components in 60% PAS-E/40% plasma (n = 8) were stored at room-temperature (20-24°C with agitation) or in the cold (2-6°C without agitation). Platelets were sampled on day 1, 5 and 14. The lipidome was assessed by ultra-pressure liquid chromatography ion mobility quadrupole time of flight mass spectrometry (UPLC IMS QToF). Changes in bioactive lipid mediators were measured by ELISA. RESULTS: The total phospholipid and sphingolipid content of the platelets and supernatant were 44 544 ± 2915 µg/mL and 38 990 ± 10 880 µg/mL, respectively, and was similar over 14 days, regardless of storage temperature. The proportion of the procoagulant lipids, phosphatidylserine (PS) and phosphatidylethanolamine (PE), increased by 2.7% and 12.2%, respectively, during extended cold storage. Cold storage for 14 days increased sphingomyelin (SM) by 4.1% and decreased ceramide by 1.6% compared to day 1. Further, lysophosphatidylcholine (LPC) species remained unchanged during cold storage for 14 days. The concentration of 12- and 15-hydroxyeicosatetraenoic acid (HETE) were lower in the supernatant of cold-stored platelets than room-temperature controls stored for 14 days. CONCLUSION: The lipid profile of platelets was relatively unchanged during storage for 5 days, regardless of temperature. However, during extended cold storage (14 days) the proportion of the procoagulant lipids, PS and PE, increased, while LPC and bioactive lipids were stable.

A deep eutectic solvent is an effective cryoprotective agent for platelets.

The most widely used method of platelet cryopreservation requires the addition of dimethyl sulfoxide (DMSO; Me2SO) as a cryoprotective agent (CPA) and pre-freeze removal of Me2SO before freezing to mitigate toxicity. However, alternative CPAs such as deep eutectic solvents (DES), which are less toxic could simplify this process. The aim of this study was to determine the effectiveness of a Proline-Glycerol (Prol-Gly 1:3) DES as a platelet CPA. Platelets were cryopreserved at -80 °C using 10 % Prol-Gly 1:3 (DES; n = 6), or in the absence of a cryoprotectant (no CPA; n = 6). Platelets were also cryopreserved according to the gold-standard blood-banking method using 5.5 % Me2SO (n = 6), with centrifugation and pre-freeze removal of the excess Me2SO. Platelet quality was assessed by flow cytometry and thromboelastography (TEG). Post-thaw recovery was similar between the three groups. The abundance of labile platelet glycoproteins GPIbα and GPVI were highest in the DES group, however, markers of activation (CD62P and annexin-V) were also higher in this group. In terms of function, the strength of the clot (maximum amplitude; TEG) and extent of clot retraction was better with DES platelets compared to no CPA, but lower than Me2SO platelets. DES provides a cryoprotective advantage to platelets when compared to no CPA. Importantly, when compared to Me2SO platelets, most quality parameters were similar in DES platelets. The major advantage with using a DES is biocompatibility, therefore it does not need to be removed prior to transfusion. This greatly simplifies the freezing and thawing process, avoiding the toxic effects of Me2SO.

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.

Risk factors for T-cell lymphopenia in frequent platelet donors: The BEST collaborative study.

BACKGROUND: Severe T-cell lymphopenia of uncertain clinical significance has been observed in frequent apheresis platelet donors. Two commonly used plateletpheresis instruments are the Trima Accel, which uses a leukoreduction system (LRS) chamber to trap leukocytes and the Fenwal Amicus, which does not use an LRS chamber. STUDY DESIGN AND METHODS: We performed an international, multicenter, observational study comparing T-cell populations in frequent platelet donors collected exclusively using the Trima instrument (n = 131) or the Amicus instrument (n = 77). Age- and sex-matched whole blood donors (n = 126) served as controls. RESULTS: CD4+ T-cell counts <200 cells/µL were found in 9.9% of frequent Trima (LRS+) platelet donors, 4.4% of frequent Amicus (LRS-) platelet donors, and 0 whole blood donors (p < .0001). CD4+ T-cell counts <200 cells/µL were only seen in platelet donors with ≥200 lifetime donations. In multivariable analysis, age, lifetime donations, and instrument (Trima vs. Amicus) were independent risk factors for lymphopenia. In 40 Trima platelet donors, a plasma rinseback procedure was routinely performed following platelet collections. No Trima platelet donors receiving plasma rinseback had a CD4+ T-cell count <200 cells/µL versus 13/91 Trima platelet donors not receiving plasma rinseback (p = .01). DISCUSSION: Recurrent bulk lymphocyte removal appears to contribute to the development of T-cell lymphopenia in frequent, long-term platelet donors. Lymphopenia is more common when an LRS chamber is used during platelet collection but can occur without an LRS chamber. Blood centers using LRS chambers can mitigate donor lymphopenia by performing plasma rinseback.

Inactivation of SARS-CoV-2 infectivity in platelet concentrates or plasma following treatment with ultraviolet C light or with methylene blue combined with visible light.

BACKGROUND: Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is unlikely to be a major transfusion-transmitted pathogen; however, convalescent plasma is a treatment option used in some regions. The risk of transfusion-transmitted infections can be minimized by implementing Pathogen Inactivation (PI), such as THERAFLEX MB-plasma and THERAFLEX UV-Platelets systems. Here we examined the capability of these PI systems to inactivate SARS-CoV-2. STUDY DESIGN AND METHODS: SARS-CoV-2 spiked plasma units were treated using the THERAFLEX MB-Plasma system in the presence of methylene blue (~0.8 µmol/L; visible light doses: 20, 40, 60, and 120 [standard] J/cm2 ). SARS-CoV-2 spiked platelet concentrates (PCs) were treated using the THERAFLEX UV-platelets system (UVC doses: 0.05, 0.10, 0.15, and 0.20 [standard] J/cm2 ). Samples were taken prior to the first and after each illumination dose, and viral infectivity was assessed using an immunoplaque assay. RESULTS: Treatment of spiked plasma with the THERAFLEX MB-Plasma system resulted in an average ≥5.03 log10 reduction in SARS-CoV-2 infectivity at one third (40 J/cm2 ) of the standard visible light dose. For the platelet concentrates (PCs), treatment with the THERAFLEX UV-Platelets system resulted in an average ≥5.18 log10 reduction in SARS-CoV-2 infectivity at the standard UVC dose (0.2 J/cm2 ). CONCLUSIONS: SARS-CoV-2 infectivity was reduced in plasma and platelets following treatment with the THERAFLEX MB-Plasma and THERAFLEX UV-Platelets systems, to the limit of detection, respectively. These PI technologies could therefore be an effective option to reduce the risk of transfusion-transmitted emerging pathogens.

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.

Eye drops of human origin-Current status and future needs: Report on the workshop organized by the ISBT Working Party for Cellular Therapies.

BACKGROUND AND OBJECTIVES: Serum eye drops (SEDs) are used to treat ocular surface disease (OSD) and to promote ocular surface renewal. However, their use and production are not standardized, and several new forms of human eye drops have been developed. MATERIALS AND METHODS: The International Society for Blood Transfusion Working Party (ISBT WP) for Cellular Therapies held a workshop to review the current types of eye drops of human origin (EDHO) status and provide guidance. RESULTS: The ISBT WP for Cellular Therapies introduced the new terminology 'EDHO' to emphasize that these products are analogous to 'medical products of human origin'. This concept encompasses their source (serum, platelet lysate, and cord blood) and the increasingly diverse spectrum of clinical usage in ophthalmology and the need for traceability. The workshop identified the wide variability in EDHO manufacturing, lack of harmonized quality and production standards, distribution issues, reimbursement schemes and regulations. EDHO use and efficacy is established for the treatment of OSD, especially for those refractory to conventional treatments. CONCLUSION: Production and distribution of single-donor donations are cumbersome and complex. The workshop participants agreed that allogeneic EDHO have advantages over autologous EDHO although more data on clinical efficacy and safety are needed. Allogeneic EDHOs enable more efficient production and, when pooled, can provide enhanced standardization for clinical consistency, provided optimal margin of virus safety is ensured. Newer products, including platelet-lysate- and cord-blood-derived EDHO, show promise and benefits over SED, but their safety and efficacy are yet to be fully established. This workshop highlighted the need for harmonization of EDHO standards and guidelines.

Increasing the time-to-freezing for clinical apheresis plasma meets quality specifications.

BACKGROUND AND OBJECTIVES: In Australia, the vast distances between blood collection centres and processing facilities make it challenging to align supply with demand. Increasing the time to freezing for clinical plasma beyond 6 h would alleviate supply issues. This study aimed to determine the quality of clinical apheresis plasma frozen within 12 h of collection. MATERIALS AND METHODS: Apheresis plasma (n = 20) collected at donor centres was immediately transported to a blood processing facility, stored at 26°C and sampled aseptically at 6, 8 and 12 h post collection. Frozen samples were thawed, and coagulation factors (F) II, V, VII, VIII and XIII, von Willebrand factor (vWF) and fibrinogen were measured using a coagulation analyser. RESULTS: FVIII concentrations declined in plasma frozen at 6, 8 and 12 h post collection (1.22 ± 0.27, 1.21 ± 0.25 and 1.16 ± 0.24 IU/mL, respectively) but not significantly (p = 0.3338). Importantly, all components met the FVIII specification (>0.7 IU/mL) for clinical plasma. Fibrinogen concentrations were stable from 6 to 12 h (p = 0.3100), as were vWF concentrations (p = 0.1281). Coagulation factors II, V, VII and XIII were not significantly different (p > 0.05 for all factors). CONCLUSION: Clinical apheresis plasma can be frozen within 12 h of collection, allowing collections from donor centres further from processing centres and increasing supply.

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.

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.

Donor citrate reactions influence the phenotype of apheresis platelets following storage.

BACKGROUND: Platelet collection and processing methods, as well as donor attributes, can influence platelet function and quality during ex vivo storage. In this study, activation and procoagulant responses in platelets collected from donors experiencing a citrate reaction (CR) were investigated. STUDY DESIGN AND METHODS: Apheresis platelet components (n = 54) were stored in 100% autologous plasma and tested on days 1 and 5 post-collection. Platelet components were categorized into two groups according to whether the donor had experienced a CR during donation (n = 10; non-CR group, n = 44). Platelet aggregation was initiated with collagen and thrombin. Platelet phenotype was characterized by flow cytometry. Fibrinogen binding was assessed following collagen + thrombin stimulation (COATed platelets), and procoagulant activity was assessed using a procoagulant phospholipid assay (PPL). Platelet microparticle (PMP) subsets were enumerated by flow cytometry. RESULTS: Basal von Willebrand factor (VWF) binding was higher in the CR donations when compared with the non-CR group. Collagen aggregation was significantly higher in platelets from CR donations, in contrast to aggregation induced by thrombin. The proportion of phosphatidylserine (PS) positive PMP and PPL clotting time were higher in the CR group, in contrast to the number of basal PS+ platelets and COATed platelets following stimulation. CONCLUSION: Platelets donated by donors who experienced a CR during donation had higher platelet activation response and possibly a more procoagulant PMP phenotype, suggesting that this donor reaction might lead to increased platelet activation.

Changes in glycans on platelet microparticles released during storage of apheresis platelets are associated with phosphatidylserine externalization and phagocytosis.

BACKGROUND: Platelets shed platelet microparticles (PMP) when activated or stored. As the removal of sialic acid (desialylation) promotes platelet uptake and clearance from the circulation, similar mechanisms for PMP uptake were hypothesized. The aim of the study was to investigate the role of surface glycans in the in vitro uptake of PMP from stored platelet components. STUDY DESIGN AND METHODS: Apheresis platelet components were stored in 40% plasma/60% SSP+ and sampled on day 1, 5, and 7 post-collection. PMP were characterized by staining with annexin-V (AnV) for phosphatidylserine (PS)-exposure, CD41 antibody, and fluorescently labeled glycan-binding lectins using flow cytometry. The procoagulant function of PMP following desialylation by neuraminidase treatment was assessed by AnV binding and a procoagulant phospholipid assay. PMP were isolated and stained with Deep Red, and phagocytosis by HepG2 cells was measured. Isolated PMP were deglycosylated with neuraminidase and galactosidase to assess the involvement of glycans in mediating phagocytosis. RESULTS: While the overall platelet surface glycan profile was unchanged during storage, PS+ platelets were sialylated, indicating different glycoproteins were changed. In contrast, sialic acid was removed from PS+ and CD41+ PMP, which specifically lost α-2,3-linked sialic acid during platelet storage. PMP were phagocytized by HepG2 cells, and PMP from platelets stored for 7 days were phagocytized to a lesser extent than on day 1. Desialylation by neuraminidase induced PS-exposure on PMP, decreased PPL clotting time, and increased PMP phagocytosis. CONCLUSION: PMP glycans change during platelet storage. Desialylation influences the procoagulant function of PMP and phagocytosis by HepG2 cells.

The compound effect of irradiation and familial pseudohyperkalemia on potassium leak from red blood cells.

BACKGROUND: Familial pseudohyperkalemia (FP) is a rare asymptomatic condition characterized by an increased rate of potassium leak from red blood cells (RBC) on refrigeration. Gamma irradiation compromises RBC membrane integrity and accelerates potassium leakage. Here, we compared the effect of irradiation, applied early or late in storage, on FP versus non-FP RBC. STUDY DESIGN: Five FP and 10 non-FP individuals from the National Institute for Health Research Cambridge BioResource, UK, and three FP and six non-FP individuals identified by Australian Red Cross Lifeblood consented to the study. Blood was collected according to standard practice in each center, held overnight at 18-24°C, leucocyte-depleted, and processed into red cell concentrates (RCC) in Saline Adenine Glucose Mannitol. On Day 1, RCC were split equally into six Red Cell Splits (RCS). Two RCS remained non-irradiated, two were irradiated on Day 1 and two were irradiated on Day 14. RBCs were tested over cold storage for quality parameters. RESULTS: As expected, non-irradiated FP RCS had significantly higher supernatant potassium levels than controls throughout 28 days of storage (p < .001). When irradiated early, FP RCS released potassium at similar rates to control. When irradiated late, FP RCS supernatants had higher initial post-irradiation potassium concentration than controls but were similar to controls by the end of storage (14 days post-irradiation). No other parameters studied showed a significant difference between FP and control. DISCUSSION: FP does not increase the rate of potassium leak from irradiated RBCs. Irradiation may cause a membrane defect similar to that in FP RBCs.

Red cells manufactured from lipaemic whole blood donations: Do they have higher haemolysis?

BACKGROUND AND OBJECTIVES: Lipaemia in blood donations is thought to influence haemolysis in stored red blood cell (RBC) components. Higher lipid concentrations are believed to increase red cell fragility, exacerbating haemolysis during collection and subsequent red cell storage. This study aimed to investigate associations between lipoproteins in plasma and haemolysis of red cells stored in saline-adenine-glucose-mannitol (SAGM). MATERIALS AND METHODS: Fifty-four plasma and matched RBCs were obtained from lipaemic whole blood donations. Plasma was tested for coagulation factors, triglycerides and cholesterol. Haemolysis, glucose, lactate, extracellular potassium, lactate dehydrogenase and adenosine triphosphate (ATP) were measured in RBC on Days 7, 21 and 42 of storage. Additionally, 20 plasma and matched RBCs from non-lipaemic donations were tested as controls. RESULTS: Lipaemic plasma had significantly higher triglyceride concentrations compared with non-lipaemic plasma. However, there was no significant difference in plasma cholesterol between the two groups. There were no significant differences in glucose, extracellular potassium or ATP concentrations in RBC from either group. There was no significant difference in haemolysis at expiry in lipaemic-derived and control RBC, with a weak correlation between haemolysis and either triglycerides or cholesterol. CONCLUSION: There was no significant difference in haemolysis in RBC manufactured from lipaemic and non-lipaemic whole blood donations when stored in SAGM; however, the proportion of RBC from lipaemic donations with higher haemolysis was greater than in the controls. There was a weak correlation between red cell haemolysis and plasma triglycerides. Therefore, RBCs derived from lipaemic donations are suitable for blood bank inventories.