Platelet lysate can support the development of a 3D-engineered skin for clinical application.

Safety concerns associated with foetal bovine serum (FBS) have restricted its translation into clinics. We hypothesised that platelet lysate (PL) can be utilised as a safe alternative to produce serum-free 3D-engineered skin. PL supported a short-term expansion of fibroblasts, with negligible replication-induced senescence and directed epidermal stratification. PL-expanded fibroblasts were phenotypically separated into three subpopulations of CD90+FAP+, CD90+FAP- and CD90-FAP+, based on CD90 (reticular marker) and FAP (papillary marker) expression profile. PL drove the expansion of the intermediate CD90+ FAP+ subpopulation in expense of reticular CD90+FAP-, which may be less fibrotic once grafted. The 3D-engineered skin cultured in PL was analysed by immunofluorescence using specific markers. Detection of ColIV and LMN-511 confirmed basement membrane. K10 confirmed near native differentiation pattern of neo-epidermis. CD29- and K5-positive interfollicular stem cells were also sustained. Transmission and scanning electron microscopies detailed the ultrastructure of the neo-dermis and neo-epidermis. To elucidate the underlying mechanism of the effect of PL on skin maturation, growth factor contents in PL were measured, and TGF-ß1 was identified as one of the most abundant. TGF-ß1 neutralising antibody reduced the number of Ki67-positive proliferative cells, suggesting TGF-ß1 plays a role in skin maturation. Moreover, the 3D-engineered skin was exposed to lucifer yellow on days 1, 3 and 5. Penetration of lucifer yellow into the skin was used as a semi-quantitative measure of improved barrier function over time. Our findings support the concept of PL as a safe and effective serum alternative for bioengineering skin for cell therapies.

Maximising platelet availability by delaying cold storage.

BACKGROUND AND OBJECTIVES: Cold-stored platelets may be an alternative to conventional room temperature (RT) storage. However, cold-stored platelets are cleared more rapidly from circulation, reducing their suitability for prophylactic transfusion. To minimise wastage, it may be beneficial to store platelets conventionally until near expiry (4 days) for prophylactic use, transferring them to refrigerated storage to facilitate an extended shelf life, reserving the platelets for the treatment of acute bleeding. MATERIALS AND METHODS: Two ABO-matched buffy-coat-derived platelets (30% plasma/70% SSP+) were pooled and split to produce matched pairs (n = 8 pairs). One unit was stored at 2-6°C without agitation (day 1 postcollection; cold); the second unit was stored at 20-24°C with constant agitation until day 4 then stored at 2-6°C thereafter (delayed-cold). All units were tested for in vitro quality periodically over 21 days. RESULTS: During storage, cold and delayed-cold platelets maintained a similar platelet count. While pH and HSR were significantly higher in delayed-cold platelets, other metabolic markers, including lactate production and glucose consumption, did not differ significantly. Furthermore, surface expression of phosphatidylserine and CD62P, release of soluble CD62P and microparticles were not significantly different, suggesting similar activation profiles. Aggregation responses of delayed-cold platelets followed the same trend as cold platelets once transferred to cold storage, gradually declining over the storage period. CONCLUSION: The metabolic and activation profile of delayed-cold platelets was similar to cold-stored platelets. These data suggest that transferring platelets to refrigerated storage when near expiry may be a viable option for maximising platelet inventories.

Refrigeration, cryopreservation and pathogen inactivation: an updated perspective on platelet storage conditions.

Conventional storage of platelet concentrates limits their shelf life to between 5 and 7 days due to the risk of bacterial proliferation and the development of the platelet storage lesion. Cold storage and cryopreservation of platelets may facilitate extension of the shelf life to weeks and years, and may also provide the benefit of being more haemostatically effective than conventionally stored platelets. Further, treatment of platelet concentrates with pathogen inactivation systems reduces bacterial contamination and provides a safeguard against the risk of emerging and re-emerging pathogens. While each of these alternative storage techniques is gaining traction individually, little work has been done to examine the effect of combining treatments in an effort to further improve product safety and minimize wastage. This review aims to discuss the benefits of alternative storage techniques and how they may be combined to alleviate the problems associated with conventional platelet storage.

Serum eye drops: a survey of international production methods.

BACKGROUND: Serum eye drops (SEDs) are used to treat dry eye syndrome and non-healing corneal lesions when other treatments fail. Despite many clinical studies demonstrating the efficacy of both autologous and allogeneic SEDs, there is no internationally harmonized method for producing SEDs. MATERIALS AND METHODS: A 40-question survey requesting information regarding donor selection, blood collection and processing, infectious disease screening, shelf life and regulatory requirements for the production of autologous and allogeneic SEDs was developed by the Biomedical Excellence for Safer Transfusion Collaborative. Survey data were collected into a database via a secure web interface and then downloaded into Excel for further analysis. RESULTS: A total of 55 responses were received, with 21 responses from centres indicating they produce SEDs. Based on the responses, collection and processing practices differ widely, according to the size of the centre making the SEDs, and their ability to collect, process and test the blood. CONCLUSION: Despite divergences in the methods for producing SEDs, the end result is a small-volume aliquot of serum that can be administered by a patient at home. If more centres move from producing autologous to allogeneic SEDs, this may provide an opportunity for production methods to become more standardized internationally.

Reduction of biological response modifiers in the supernatant of washed paediatric red blood cells.

BACKGROUND: Washing of red blood cells (RBC) can reduce unwanted biological response modifiers (BRMs) that can mediate transfusion complications in infants. The aim of this study was to examine the in vitro quality and the changes in BRMs following washing in paediatric RBC units. MATERIALS AND METHODS: A pool and split design was used to prepare RBC (either 1 or 4 days old; n = 26 pairs). One unit was washed with 0·9% saline by centrifugation and then resuspended in SAG-M, while the other remained unwashed. Each RBC unit was divided to produce four units of paediatric-sized components. Samples were taken after 3 h and subsequently on days 1, 2, 7 and 14 post-wash. RESULTS: Washing of RBC resulted in some red cell loss, with a minor increase in haemolysis. Washing effectively reduced supernatant potassium and IgA, as well as cytokines and complement proteins. RBC microparticles were significantly reduced in RBC washed at 1, but not 4 days post-collection. Incubation with supernatant from unwashed but not washed RBC led to endothelial cell activation, with increased cell surface expression of CD62E (E-selectin) and CD106 (VCAM). CONCLUSION: Although washing affected some aspects of the in vitro quality of RBC, it effectively reduced the concentration and activity of BRMs in the supernatant of RBC. Such a reduction may be clinically beneficial in selected patient groups.

Riboflavin and ultraviolet light: impact on dengue virus infectivity.

BACKGROUND: Dengue viruses (DENV 1-4) are emerging across the world, and these viruses pose a risk to transfusion safety. Pathogen inactivation may be an alternative approach for managing the risk of DENV transfusion transmission. This study aimed to investigate the ability of riboflavin and UV light to inactivate DENV 1-4 in platelet concentrates. MATERIALS AND METHODS: DENV 1-4 were spiked into buffy coat-derived platelet concentrates in additive solution (SSP+) before being treated with riboflavin and UV light. Infectious virus was quantified pre- and posttreatment, and the reduction in viral infectivity was calculated. RESULTS: All four DENV serotypes were modestly reduced after treatment. The greatest amount of reduction in infectivity was observed for DENV-4 (1·81 log reduction) followed by DENV-3 (1·71 log reduction), DENV-2 (1·45 log reduction) and then DENV-1 (1·28 log reduction). CONCLUSION: Our study demonstrates that DENV 1-4 titres are modestly reduced following treatment with riboflavin and UV light. With the increasing number of transfusion-transmitted cases of DENV around the globe, and the increasing incidence and geographical distribution of DENV, additional approaches for maintaining blood safety may be required in the future.

Gamma-irradiation of deglycerolized red cells does not significantly affect in vitro quality.

BACKGROUND AND OBJECTIVES: Red cells frozen with glycerol may require gamma-irradiation after thawing and deglycerolization for transfusion to at-risk patients. Both freezing and irradiation are known to cause red cell damage. However, the effect of irradiation on the quality of deglycerolized red cells and the optimal shelf life of such a component is currently unknown. MATERIALS AND METHODS: Red cells (<7 days) were pooled, split and glycerolized using an ACP-215 automated cell washer (n = 12 pairs) and frozen at -80°C. Red cells were thawed, deglycerolized and resuspended in SAG-M. One of each pair was gamma-irradiated, while the other served as a control. Products were stored at 2-6°C and sampled for in vitro testing immediately after irradiation, and at 24 and 48 h postirradiation. RESULTS: Irradiation of deglycerolized red cells led to a >1·5-fold increase in extracellular potassium, compared to control units at 24 and 48 h postirradiation. Other parameters, including haemolysis, were not significantly affected by irradiation postdeglycerolization. CONCLUSION: Deglycerolized, irradiated red cells had increased supernatant potassium, but remained of acceptable quality for 24 h postirradiation.

Treatment of platelets with riboflavin and ultraviolet light mediates complement activation and suppresses monocyte interleukin-12 production in whole blood.

BACKGROUND AND OBJECTIVES: Pathogen inactivation (PI) and storage may alter the immunomodulatory capacity of platelets (PLTs). The aim of this study was to examine the effect of PI (Riboflavin and ultraviolet light treatment) and storage on the capacity of PLTs to induce cytokine responses in recipient inflammatory cells. MATERIALS AND METHODS: A pool and split design was used to prepare untreated and PI-treated buffy coat-derived platelet concentrates (PCs). Samples were taken on days 2 and 7 postcollection and incubated with ABO/RhD-matched fresh whole blood for 6 h with or without lipopolysaccharide (LPS). The intracellular production of IP-10, MCP-1, MIP-1α, IL-8, IL-6, IL-10, IL-12, TNF-α and MIP-1ß in monocytes and neutrophils was assessed using flow cytometry. Complement proteins in PLT supernatants were measured using a cytometric bead array. RESULTS: PLTs and PLT supernatant (both untreated and PI-treated) resulted in modulation of intracellular MIP-1ß and IL-12 production in monocytes. Compared to untreated PLTs, PI-treated PLTs resulted in significantly lower LPS-induced monocyte IL-12 production (day 7). The concentration of C3a and C5a (and their desArg forms) was significantly increased in PLT supernatants following PI. CONCLUSION: PI results in decreased LPS-induced monocyte IL-12 production and increased complement activation. The association between platelet-induced complement activation and IL-12 production warrants further investigation.

Understanding the effects of gamma-irradiation on potassium levels in red cell concentrates stored in SAG-M for neonatal red cell transfusion.

BACKGROUND AND OBJECTIVES: Red cell transfusions, to paediatric patients, are often gamma-irradiated to prevent transfusion-associated graft-versus-host disease. This study measured changes in potassium and other in vitro parameters immediately following gamma-irradiation of paediatric and full-size red cell concentrates (RCCs). MATERIALS AND METHODS: The effects of irradiation on potassium release in RCCs stored in SAG-M were investigated under three scenarios. In the first scenario, RCC < 5 days was split into paediatric packs, gamma-irradiated and tested for potassium and haemolysis at 0, 2, 4, 6, 24 and 48 h. In the second scenario, full-size RCCs < 5 days postcollection were gamma-irradiated and tested as for the paediatric packs. Thirdly, RCCs < 14 days postcollection were gamma-irradiated and assessed at 6 and 24 h and 7 and 14 days. Each group contained paired controls that were not gamma-irradiated. RESULTS: In all situations, gamma-irradiation resulted in a twofold increase in potassium concentrations after 24 h of storage, compared to matched unirradiated controls. This difference was detectable as early as 2 h postirradiation. Few differences were observed between control and irradiated RCCs in other key parameters, including ATP, 2,3-DPG, haemoglobin, pH, glucose and lactate concentration. CONCLUSION: Gamma-irradiation of RCCs significantly increased extracellular potassium. Irradiation of fresher RCCs results in lower potassium concentrations, which is less likely to lead to hyperkalaemia upon transfusion.

Preparation of red blood cell concentrates and plasma units from whole blood held overnight using a hollow-fibre separation system.

BACKGROUND: The ErySep system represents an alternative to centrifuge-based whole blood (WB) separation, using gravity and filtration through hollow-fibres (0·2 µm pore size) to produce red blood cell (RBC) and plasma components. The aim of this study was to characterise the quality of ErySep RBC and plasma units compared with standard products from WB held overnight. METHODS/MATERIALS: Two ABO-compatible WB units (n = 24) were pooled and split to produce matched products. One of the WB units was separated into components using the ErySep system (ErySep; n = 12), whereas the other units were separated by centrifugation (control; n = 12). RBC units were stored at 2-6 °C and assessed for in vitro quality over 42 days of storage. Plasma was frozen at -30 °C and tested upon thawing. RESULTS: Processing WB with the ErySep system took longer than controls. The ErySep RBC units were of an appropriate volume (307 ± 17 mL) and contained sufficient Hb (50 ± 2 g unit(-1) ). ErySep RBC components contained more microparticles relative to controls at expiry. The plasma volume, total protein, coagulation factor activity (fibrinogen, FV, FVIII) and number of microparticles was lower in the ErySep units compared with controls. CONCLUSION: Following overnight hold of WB, the ErySep system was capable of producing RBC components that met specifications. However, the ErySep plasma components did not meet quality specifications.

Evaluation of the quality of blood components prepared using the Reveos automated blood processing system.

BACKGROUND AND OBJECTIVE: The Reveos automated blood processing system has been developed to combine primary and secondary processing of whole-blood units, resulting in a plasma unit, a red-blood-cell concentrate and an interim platelet unit per input. The aim of this study was to determine product specifications and in vitro quality of components produced by the Reveos system. MATERIALS AND METHODS: Whole blood was processed using the Reveos system and compared with historical Reference units produced using semi-automated methods. Reveos red cells were leucoreduced and stored in SAGM at 4°C. Reveos plasma was frozen at -30°C and factor activity was assessed after thawing. Reference red cell, plasma and buffy coats were produced by top and bottom processing. Leucoreduced Reveos and Reference platelet concentrates were prepared by pooling four interim platelet units or four buffy coats, respectively, with SSP+. RESULTS: Processing with the Reveos system was faster (76 min) than semi-automated separation (92 min). The red cell and platelet yields were higher in the units prepared by the Reveos system. The Reference and Reveos red cell and plasma units had very similar in vitro quality parameters. The platelet concentrates were also similar in many in vitro parameters, including pH, glucose and lactate metabolism, hypotonic shock response and phosphatidylserine expression, although platelet activation markers (CD62P and cytokine levels) were higher in the Reveos units. CONCLUSION: The Reveos system can improve blood component efficiencies through reductions in processing time, whilst maintaining similar component quality.

In vitro assessment of buffy-coat derived platelet components suspended in SSP+ treated with the INTERCEPT Blood system.

BACKGROUND: The INTERCEPT Blood System uses amotosalen-HCl and UVA light to cross-link DNA and RNA, thereby inhibiting pathogen replication. Although previous studies have shown that this treatment alters in vitro platelet quality, most studies have assessed apheresis platelets or platelets pooled from 5 or 6 donors. In Australia, platelets are prepared using buffy-coats from 4 donors, with SSP+ and have lower plasma carryover than recommended by the manufacturer (32-47%). As such, it is currently unknown whether these platelet concentrates are suitable for INTERCEPT treatment. MATERIALS AND METHODS: Platelet concentrates were prepared by pooling four buffy-coats with SSP+, resulting in 30% plasma carryover. Two platelet units were pooled and split to generate paired units, with one unit treated with the INTERCEPT System (n = 6), whilst the other remained untreated (n = 6). All units were stored for seven days at 22 °C with agitation. RESULTS: INTERCEPT treatment resulted in 10·4 ± 4·3% loss of platelets, but did not significantly affect the functional integrity of mitochondria. INTERCEPT-treated platelets demonstrated a decreased pH, accelerated lactate production and glucose consumption, as well as higher surface expression and increased secretion of P-selectin and reduced collagen-induced aggregation. These changes were particularly evident from day 5 of storage. CONCLUSION: The observed increase in platelet glycolysis following INTERCEPT treatment is consistent with previous literature reports. Importantly, the in vitro changes were less marked than previously reported indicating that the platelets suspended in SSP+ with reduced plasma carryover are of suitable in vitro quality following INTERCEPT treatment and storage.

The effect of pathogen reduction technology (Mirasol) on platelet quality when treated in additive solution with low plasma carryover.

BACKGROUND AND OBJECTIVES: Pathogen reduction technologies (PRT) for platelets are now compatible with both plasma and platelet additive solutions (PAS). The aim of this study was to examine the effect of PRT on the platelet storage lesion, in the presence of PAS with low plasma carryover. MATERIALS AND METHODS: PRT-treated (Mirasol) and untreated buffy coat-derived platelet concentrates prepared in 28% plasma/PAS-IIIM were evaluated using in vitro cell quality parameters on days 1, 2, 5, and 7 post-collection. RESULTS: At day 5, there were no significant differences between control and PRT treated platelets for swirl, viability, pO(2) , pCO(2) , mean platelet volume and adenosine diphosphate-induced aggregation. PRT treatment did not affect the functional integrity of the mitochondria. However, PRT resulted in a decrease in pH and enhancement of platelet glycolysis and activation, evidenced by increased glucose consumption and lactate production rates, increased expression of CD62P, CD63, annexin V staining and increased secretion of cytokines (P < 0.05). Hypotonic shock response and aggregation in response to collagen were also significantly reduced in PRT treated platelets (P < 0.05). CONCLUSION: Despite the observed differences in platelet metabolism and activation observed following PRT treatment in PAS and low plasma carryover, the results suggest that treatment and storage of platelets in PAS is no more detrimental to platelets than treatment and storage in plasma.

Cryopreservation of buffy-coat-derived platelet concentrates in dimethyl sulfoxide and platelet additive solution.

Platelets prepared in plasma can be frozen in 6% dimethyl sulfoxide (Me(2)SO) and stored for extended periods at -80°C. The aim of this study was to reduce the plasma present in the cryopreserved product, by substituting plasma with platelet additive solution (PAS; SSP+), whilst maintaining in vitro platelet quality. Buffy coat-derived pooled leukoreduced platelet concentrates were frozen in a mixture of SSP+, plasma and 6% Me(2)SO. The platelets were concentrated, to avoid post-thaw washing, and frozen at -80°C. The cryopreserved platelet units (n=9) were rapidly thawed at 37°C, reconstituted in 50% SSP+/plasma and stored at 22°C. Platelet recovery and quality were examined 1 and 24h post-thaw and compared to the pre-freeze samples. Upon thawing, platelet recovery ranged from 60% to 80%. However, there were differences between frozen and liquid-stored platelets, including a reduction in aggregation in response to ADP and collagen; increased CD62P expression; decreased viability; increased apoptosis and some loss of mitochondrial membrane integrity. Some recovery of these parameters was detected at 24h post-thaw, indicating an extended shelf-life may be possible. The data suggests that freezing platelets in 6% Me(2)SO and additive solution produces acceptable in vitro platelet quality.

A platelet-derived hydrogel improves neovascularisation in full thickness wounds.

Platelets are a reservoir of growth factors, cytokines and chemokines involved in spontaneous wound repair. In this study, a platelet-rich and fibrin-rich hydrogel was generated from expired platelet components that would have otherwise been transfused. The material contained physiological concentrations of transforming growth factor ß1 (TGF-ß1, platelet-derived growth factor AB (PDGF-AB), PDGF-BB, insulin-like growth factor-1 (IGF-1), fibroblast growth factor 2 (FGF-2), and epidermal growth factor (EGF). The effect of the hydrogel on wound repair was investigated in SKH-1 mice. Full thickness dorsal wounds were created on the mice and treated with the hydrogel at various concentrations. Immunohistochemical staining with CD31 (endothelial cell marker) revealed that wounds treated with the hydrogel showed significantly enhanced vascularisation in the wound bed. Moreover, high levels of interleukin-6 (IL-6) and KC (IL-8 functional homologue) in treated wounds were sustained over a longer period of time, compared to untreated wounds. We postulate that sustained IL-6 is a driver, at least partly, of enhanced vascularisation in full thickness wounds treated with the hydrogel. Future work is needed to explore whether this hydrogel can be utilised as a treatment option when vascularisation is a critical limitation. STATEMENT OF SIGNIFICANCE: The economic cost of wound repair is estimated in billions of dollars each year. In many cases time required to vascularise wounds is a major contributor to slow wound repair. In this study, we developed a blood-derived platelet- and fibrin-rich hydrogel. It contains a number of growth factors actively involved in spontaneous wound healing. This hydrogel significantly improved dermal repair and vascularisation in a full-thickness wound mouse model. This study provides an action mechanism for modulation of localised inflammation.