In vitro characteristics and in vivo platelet quality of whole blood treated with riboflavin and UVA/UVB light and stored for 24 hours at room temperature.

BACKGROUND: There is a global increase in whole blood usage and at the same time, emerging pathogens give cause for pathogen reduction technology (PRT). The Mirasol PRT has shown promising results for plasma and platelet concentrate products. Treatment of whole blood with subsequent platelet survival and recovery analysis would be of global value. STUDY DESIGN AND METHODS: A two-arm, open-label laboratory study was performed with 40 whole blood collections in four groups: non-leukoreduced non-PRT-treated, non-leukoreduced PRT-treated, leukoreduced non-PRT-treated, and leukoreduced PRT-treated. Leukoreduction and/or PRT-treatment was performed on the day of collection, then all WB units were stored at room temperature for 24 h. Sampling was performed after hold-time and after 24-h storage in RT. If PRT-treatment or leukoreduction, samples were also taken subsequently after treatment. Thirteen healthy volunteer blood donors completed the in vivo study per protocol. All WB units were non-leukoreduced and PRT-treated. Radioactive labeling of platelets from RT-stored, PRT-treated whole blood, sampling of subjects, recovery, and survival calculations were performed according to the Biomedical Excellence for Safer Transfusion Collaborative protocol. RESULTS: In vitro characteristics show that PRT-treatment leads to increased levels of hemolysis, potassium, and lactate, while there are decreased levels of glucose, FVIII, and fibrinogen after 24 h of storage. All values are within requirements for WB. In vivo recovery and survival of platelets were 85.4% and 81.3% of untreated fresh control, respectively. CONCLUSIONS: PRT-treatment moderately reduces whole blood quality but is well within the limits of international guidelines. Recovery and survival of platelets are satisfactory after Mirasol treatment.

Cryopreservation of platelets treated with riboflavin and UV light and stored at -80°C for 1 year.

BACKGROUND: The combination of pathogen reduction technologies (PRTs) and cryopreservation can contribute to building a safe and durable platelet (PLT) inventory. Information about cryopreserved riboflavin and UV light-treated PLTs is scarce. STUDY DESIGN AND METHODS: Twenty-four buffy coat (BC) PLT concentrates were grouped into 12 type-matched pairs, pooled, and divided into 12 non-PRT-treated control units and 12 riboflavin and UV light PRT-treated test units. Both were cryopreserved with 5% DMSO and stored at -80°C for 1 year. The cryopreservation method used was designed to avoid the formation of aggregates. PLT variables (PLT recovery, swirling, pH, MPV, and LDH) and hemostatic function measured by thromboelastography (TEG) were analyzed before cryopreservation (day 1) and post-cryopreservation at day 14 and months 3, 6, and 12 of storage at -80°C. The analyses were carried out within 1-h post-thaw. RESULTS: No aggregates were found in either PLT group at any time. Swirling was observed in both groups. MPV increased and mean pH values decreased over time (p < .001), but the mean pH value was never below 6.4 in either group after 12 months of storage at -80°C. PLT recovery was good and clotting time became significantly shorter over the storage period in both groups (p < .001). CONCLUSION: Our cryopreservation and thawing method prevented aggregate formation in cryopreserved riboflavin-UV-light-treated PLTs, which exhibited good recovery, swirling, pH > 6.4, and procoagulant potential, as evidenced by a reduced clotting time after 12 months of storage at -80°C. The clinical relevance of these findings should be further investigated in clinical trials.

The effect of gamma irradiation on platelet redox state during storage.

BACKGROUND: As a method with insignificant adverse effects on in vitro quality of platelet concentrates (PCs), gamma irradiation is applied to abrogate the risk of transfusion-associated graft-vs-host disease in vulnerable recipients. However, there is some evidence of lower posttransfusion responses and proteomic alterations in gamma-irradiated platelets (PLTs), which raises some questions about their quality, safety, and efficacy. Since reactive oxygen species (ROS) are considered as markers of PLT storage lesion (PSL), the study presented here investigated oxidant state in gamma-irradiated PCs. STUDY DESIGN AND METHODS: PLT-rich plasma PC was split into two bags, one kept as control while other was subjected to gamma irradiation. Within 7 days of storage, the levels of intra-PLT superoxide, H2 O2 , mitochondrial ROS, P-selectin expression, and phosphatidylserine (PS) exposure were detected by flow cytometry while intracellular reduced glutathione (GSH), glucose concentration, and lactate dehydrogenase (LDH) activity were measured by enzymocolorimetric method. RESULTS: GSH decreased, while ROS generation and LDH activity increased, during storage. Gamma irradiation significantly attenuated GSH whereas increased ROS generation in earlier and later stages of storage associated with either P-selectin or PS exposure increments. CONCLUSION: Gamma irradiation can significantly increase cytosolic ROS generation in two distinct phases, one upon irradiation and another later in longer-stored PCs. While earlier ROS influx seems to be governed by direct effect of irradiation, the second phase of oxidant stress is presumably due to the storage-dependent PLT activation. Intriguingly, these observations were also in line with early P-selectin increments and increased PS exposure in longer-stored PLTs. Given the mutual link between ROS generation and PLT activation, further investigation is required to explore the effect of gamma irradiation on the induction of PSL.

Releasates of riboflavin/UV-treated platelets: Microvesicles suppress cytokine-mediated endothelial cell migration/proliferation.

BACKGROUND: Accelerated development of the platelet (PLT) storage lesion upon pathogen inactivation (PI) is associated with the release of proteins from granules and platelet microvesicles (PMVs). Whether PI treatments alter the interaction between PLT factors and the vessel endothelium is of interest in understanding the risk profile of these technologies. STUDY DESIGN AND METHODS: In a pool-and-split study, one platelet concentrate (PC) was treated with riboflavin/UV (RF/UV) light, while the other one was kept as an untreated control. Releasates and PMV-depleted releasates were prepared by differential centrifugation steps on days 0, 1, 5, and 7 of storage. Cytokine/chemokine release following PI treatment was analyzed by an antibody array, and results were verified by the enzyme-linked immunosorbent assay. PMVs were enumerated by CD41 labeling and flow cytometry. Wound scratch assays were performed using cultured Ea.hy926 cells exposed to the differently prepared releasates. Effects of releasates on the phosphorylation levels of kinases ERK and p38 expressed by endothelial cells were analyzed by immunoblot. RESULTS: Cytokine/chemokine assays identified a 2-fold increase in epidermal growth factor released from PCs treated with RF/UV light compared with control. PMV count increased ~100-fold following PI treatment. Unmodified releasates and PMV-depleted releasates displayed different contributions to the kinetics of endothelial cell wound closure. This observation was associated with an increased ERK versus unaltered p38 activation in the endothelial cells. CONCLUSION: This study identified an inhibitory impact of PMVs on endothelial cell migration/proliferation upon stimulation by released cytokines and PMVs from PLTs treated with RF/UV light for endothelial cell wound closure.

Evaluation of riboflavin and ultraviolet light treatment against Klebsiella pneumoniae in whole blood-derived platelets: A pilot study.

BACKGROUND: Bacterial contamination of platelet concentrates (PCs) is the predominant cause of infectious transfusion reactions. The Pathogen Inactivation Mirasol system was implemented at the King Faisal Specialist Hospital (Saudi Arabia) to reduce the risk of transfusing contaminated PCs. This pilot study evaluated the effectiveness of Mirasol against Klebsiella pneumoniae, a pathogen associated with transfusion reactions, in whole blood-derived PCs. STUDY DESIGN AND METHODS: Whole blood (WB) units inoculated with one of six K. pneumoniae strains (five clinical isolates and ATCC-700603) at a concentration of 3-38 CFU/unit, were processed using the platelet-rich plasma (PRP) method. Each spiked PC was pooled with four unspiked units. The pooled PC was split into three Mirasol storage bags: an untreated unit (control), and two units treated with Mirasol at 26 and 32 h post-WB collection, respectively. PC samples obtained before and after Mirasol treatment were used for BacT/ALERT cultures and determination of bacteria quantification. Each experiment was repeated three independent times. RESULTS: Five strains were detected prior to PC treatment (24 h post-WB spiking), while one clinical isolate was not detected. Mirasol treatment after 26 h of WB collection resulted in complete inactivation of all K. pneumoniae strains. However, treatment 32 h post-WB collection resulted in the breakthrough of one clinical isolate in two of the three replicates with ~7.8 log10 CFU/unit detected on day 5 of PC storage. CONCLUSION: Delayed Mirasol treatment from 26 to 32 h post-WB collection, resulted in one breakthrough. These results highlight the importance of minimizing the time between WB collection and PI treatment.

Effects of Intercept pathogen reduction treatment on extended cold storage of apheresis platelets.

BACKGROUND: Platelets pose the greatest transfusion-transmitted infectious risk among blood products. Refrigeration of platelets can mitigate bacterial contamination and extend platelet shelf life. Implementation of pathogen reduction technologies (PRTs) at blood banks has become increasingly popular to protect against emerging and reemerging infectious diseases. In this study, we sought to evaluate the effects of Intercept PRT on platelets collected on different platforms and cold-stored for up to 21 days in plasma and platelet additive solution (PAS). METHODS: Double-dose apheresis platelets were collected with use of a Trima or Amicus system into either 100% plasma or 65% InterSol PAS/35% plasma and split equally between two bags. One bag served as control, while the other received Intercept PRT treatment. Bags were stored unagitated in the cold and evaluated on Days 1, 7, 14, and 21 to assess platelet metabolism, activation, aggregation, and clot formation and retraction. RESULTS: By Day 14 of storage, lactate levels reached approximately 13 mmol/L for all samples irrespective of Intercept treatment. Mean clot firmness dropped from the 62.2- to 67.5-mm range (Day 1) to the 28.4- to 51.3-mm range (Day 21), with no differences observed between groups. Clot weights of Intercept-treated Trima/plasma samples were significantly higher than control by Day 14 of storage (P = .004), indicating a reduced clot retraction function. Intercept treatment caused a higher incidence of plasma membrane breakdown in plasma-stored platelets (P = .0013; Trima/plasma Day 14 Control vs Intercept). CONCLUSIONS: Intercept treatment of platelets and subsequent cold storage, in plasma or PAS, results in comparable platelet metabolism platelets for up to 14 days of storage but altered clotting dynamics. Pathogen-reduced platelets with an extended shelf life would be beneficial for the deployed setting and would greatly impact transfusion practice among civilian transfusion centers.

HLA class I depletion by citric acid, and irradiation of apheresis platelets for transfusion of refractory patients.

BACKGROUND: Patients can form antibodies to foreign human leukocyte antigen (HLA) Class I antigens after exposure to allogeneic cells. These anti-HLA class I antibodies can bind transfused platelets (PLTs) and mediate their destruction, thus leading to PLT refractoriness. Patients with PLT refractoriness need HLA-matched PLTs, which require expensive HLA typing of donors, antibody analyses of patient sera and/or crossmatching. An alternative approach is to reduce PLT HLA Class I expression using a brief incubation in citric acid on ice at low pH. METHODS AND MATERIALS: Apheresis PLT concentrates were depleted of HLA Class I complexes by 5 minutes incubation in ice-cold citric acid, at pH 3.0. Surface expression of HLA Class I complexes, CD62P, CD63, phosphatidylserine, and complement factor C3c was analyzed by flow cytometry. PLT functionality was tested by thromboelastography (TEG). RESULTS: Acid treatment reduced the expression of HLA Class I complexes by 71% and potential for C3c binding by 11.5-fold compared to untreated PLTs. Acid-treated PLTs were significantly more activated than untreated PLTs, but irrespective of this increase in steady-state activation, CD62P and CD63 were strongly upregulated on both acid-treated and untreated PLTs after stimulation with thrombin receptor agonist peptide. Acid treatment did not induce apoptosis over time. X-ray irradiation did not significantly influence the expression of HLA Class I complexes, CD62P, CD63, and TEG variables on acid treated PLTs. CONCLUSION: The relatively simple acid stripping method can be used with irradiated apheresis PLTs and may prevent transfusion-associated HLA sensitization and overcome PLT refractoriness.

In vitro thrombus formation and in vivo hemostasis mediated by platelets irradiated with bactericidal ultraviolet C from xenon flash under flow conditions.

BACKGROUND: We previously reported a flow path-ultraviolet C (UVC) irradiation system for platelet concentrates (PCs) with platelet additive solution (PAS) to minimize contamination by bacteria. Here, we investigated functionalities of irradiated platelets (PLTs) in in vitro thrombus formation and in vivo hemostasis. STUDY DESIGN AND METHODS: PAS-PCs were irradiated with flash UVC using the flow path system. Their variables (PLT count, mean platelet volume, pH, glucose, lactate, glycoprotein [GP] Ib, and activated integrin αIIbß3) were evaluated. Static adhesion to collagen or fibrinogen was analyzed using fluorescent microscopy. Thrombus formation under flow conditions was assessed using a collagen-coated bead column. Adenosine diphosphate (ADP)-induced Akt phosphorylation was determined by western blot. In vivo hemostasis and circulatory survival of PLTs were assessed with a rabbit bleeding model. RESULTS: All variables, except for GPIb expression, were slightly, but significantly, impaired after flash UVC irradiation throughout the 6-day storage period. No difference was observed in static adhesion to either collagen or fibrinogen between irradiated and nonirradiated PAS-PCs. In vitro thrombus formation of flash UVC-irradiated PAS-PCs was significantly greater than that of nonirradiated PAS-PCs. ADP-induced Akt phosphorylation was enhanced in irradiated PAS-PCs. In vivo hemostatic efficacy was comparable between the groups on Day 1. The efficacy declined in nonirradiated PAS-PCs on Day 5, while it was retained in flash UVC-irradiated PAS-PCs. Circulatory survival of PLTs was lower in irradiated PAS-PCs. CONCLUSIONS: PAS-PCs irradiated with UVC from xenon flash have favorable properties to achieve hemostasis compared with nonirradiated PAS-PCs.

Effects of nitrite and far-red light on coagulation.

Nitric oxide, NO, has been explored as a therapeutic agent to treat thrombosis. In particular, NO has potential in treating mechanical device-associated thrombosis due to its ability to reduce platelet activation and due to the central role of platelet activation and adhesion in device thrombosis. Nitrite is a unique NO donor that reduces platelet activation in that it's activity requires the presence of red blood cells whereas NO activity of other NO donors is blunted by red blood cells. Interestingly, we have previously shown that red blood cell mediated inhibition of platelet activation by adenosine diphosophate (ADP) is dramatically enhanced by illumination with far-red light that is likely due to photolysis of red cell surface bound NO congeners. We now report the effects of nitrite, far-red light, and their combination on several measure of blood coagulation using a variety of agonists. We employed turbidity assays in platelet rich plasma, platelet activation using flow cytometry analysis of a fluorescently labeled antibody to the activated platelet fibrinogen binding site, multiplate impedance-based platelet aggregometry, and assessment of platelet adhesion to collagen coated flow-through microslides. In all cases, the combination of far-red light and nitrite treatment decreased measures of coagulation, but in some cases mono-treatment with nitrite or light alone had no effect. Perhaps most relevant to device thrombosis, we observed that platelet adhesions was inhibited by the combination of nitrite and light treatment while nitrite alone and far-red light alone trended to decrease adhesion, but the results were mixed. These results support the potential of combined far-red light and nitrite treatment for preventing thrombosis in extra-corporeal or shallow-tissue depth devices where the far-red light can penetrate. Such a combined treatment could be advantageous due to the localized treatment afforded by far-red light illumination with minimal systemic effects. Given the role of thrombosis in COVID 19, application to treatment of patients infected with SARS Cov-2 might also be considered.

Antioxidant power measurement in platelet concentrates treated by two pathogen inactivation systems in different blood centres.

BACKGROUND AND OBJECTIVES: The antioxidant power measurement can be useful to validate the execution of the pathogen inactivation treatment of platelet concentrates. The aim of this study is to evaluate the technology on different blood preparations including INTERCEPT and Mirasol treatments that are in routine use in Belgium and Luxemburg. MATERIALS AND METHODS: The antioxidant power measurement was tested on 78 apheresis platelet concentrates and 54 pools of buffy-coats-derived platelet concentrates before and after INTERCEPT treatment. In addition, 100 Reveos platelet pools were tested before and after Mirasol treatment. The antioxidant power was quantified electrochemically using disposable devices and was expressed as equivalent ascorbic acid concentration. RESULTS: Mean results for apheresis platelet concentrates were of 90 ± 14 and 35 ± 10 µmol/l eq. ascorbic acid before and after INTERCEPT treatment, respectively. The mean results for pools of buffy-coats-derived platelet concentrates were of 81 ± 10 and 29 ± 4 eq. µmol/l ascorbic acid before and after INTERCEPT treatment, respectively. For buffy-coats-derived platelet concentrates treated by Mirasol technology, the mean results were of 98 ± 11 and 32 ± 10 µmol/l eq. ascorbic acid before and after illumination, respectively. CONCLUSION: The antioxidant power significantly decreases with pathogen inactivation treatments for platelet concentrates treated by INTERCEPT or Mirasol technologies.

Effects of exposure to low-dose ionizing radiation on changing platelets: a prospective cohort study.

BACKGROUND: Numerous studies have concentrated on high-dose radiation exposed accidentally or through therapy, and few involve low-dose occupational exposure, to investigate the correlation between low-dose ionizing radiation and changing hematological parameters among medical workers. METHODS: Using a prospective cohort study design, we collected health examination reports and personal dose monitoring data from medical workers and used Poisson regression and restricted cubic spline models to assess the correlation between changing hematological parameters and cumulative radiation dose and determine the dose-response relationship. RESULTS: We observed that changing platelet of 1265 medical workers followed up was statistically different among the cumulative dose groups (P = 0.010). Although the linear trend tested was not statistically significant (Ptrend = 0.258), the non-linear trend tested was statistically significant (Pnon-linear = 0.007). Overall, there was a correlation between changing platelets and cumulative radiation dose (a change of ßa 0.008 × 109/L during biennially after adjusting for gender, age at baseline, service at baseline, occupation, medical level, and smoking habits; 95% confidence interval [CI] = 0.003,0.014 × 109/L). Moreover, we also found positive first and then negative dose-response relationships between cumulative radiation dose and changing platelets by restricted cubic spline models, while there were negative patterns of the baseline service not less than 10 years (- 0.015 × 109/L, 95% CI = - 0.024, - 0.007 × 109/L) and radiation nurses(- 0.033 × 109/L, 95% CI = - 0.049, - 0.016 × 109/L). CONCLUSION: We concluded that although the exposure dose was below the limit, medical workers exposed to low-dose ionizing radiation for a short period of time might have increased first and then decreased platelets, and there was a dose-response relationship between the cumulative radiation dose and platelets changing.

Hypersensitivity transfusion reactions to platelet concentrate: a retrospective analysis of the French hemovigilance network.

BACKGROUND: Among labile blood products, platelet concentrates (PCs) are the leading cause of hypersensitivity transfusion reactions (HTRs). These reactions often lead to interruption of PC transfusion and can result in a prolonged transfusion process leading to significant morbidity and use of premedication and close monitoring for patients with a history of allergic transfusion reactions. The French hemovigilance database is one of the largest standardized databases providing information on HTRs following administration of labile blood products. In this study, we analyzed this database to assess the relative risk of HTR for each type of PC. STUDY DESIGN AND METHODS: HTRs following PC transfusion were retrospectively extracted from the e-Fit Hemovigilance database of the French National Agency for Medicines and Health Products Safety (ANSM). Frequencies were calculated using the number of specific PCs transfused. RESULTS: Between 2008 and 2014, the overall estimated incidence of HTRs following PC administration was calculated at 232 HTRs per 100,000 PCs transfused. The rate of HTRs was significantly higher with apheresis PC (337/100,000) than with buffy-coat PC (94/100,000). Platelets in additive solutions (PAS) were associated with a significantly lower frequency of HTRs when compared with PCs in native plasma. Amotosalen/UVA- PCs (APCs and BCPCs) which are always in PAS in France, exhibited the lowest frequency of HTRs when compared with their corresponding PCs in native plasma or in PAS (p < 10-7 in all comparisons). CONCLUSION: Our results showed that the type of PC and its processing may have an impact on the risk of HTR.

Inactivation of yellow fever virus with amotosalen and ultraviolet A light pathogen-reduction technology.

BACKGROUND: The reemergence of yellow fever virus (YFV) in Africa and Brazil, and massive vaccine campaigns triggered to contain the outbreaks, have raised concerns over blood transfusion safety and availability with increased risk of YFV transfusion-transmitted infections (TTIs) by native and vaccine-acquired YFV. Blood donor deferral for 2 to 4 weeks following live attenuated YFV vaccination, and deferral for travel to endemic/epidemic areas, may result in blood donor loss and impact platelet component (PC) stocks. This study investigated the efficacy of INTERCEPT Blood System pathogen reduction (PR) with use of amotosalen and ultraviolet A (UVA) light to inactivate high levels of YFV in PCs. MATERIALS: Four units of apheresis platelets prepared in 35% plasma/65% platelet additive solution (PC-PAS) and 4 units of PC in 100% human plasma (PC-Plasma) were spiked with high infectious titers of YFV (YFV-17D vaccine strain). YFV-17D infectious titers were measured by plaque assay and expressed as plaque-forming units (PFU) before and after amotosalen/UVA treatment to determine log reduction. RESULTS: The mean YFV-17D infectious titers in PC before inactivation were 5.5 ± 0.1 log PFU/mL in PC-PAS and 5.3 ± 0.1 log PFU/mL in PC-Plasma. No infectivity was detected immediately after amotosalen/UVA treatment. CONCLUSION: The amotosalen/UVA PR system inactivated high titers of infectious YFV-17D in PC. This PR technology could reduce the risk of YFV TTI and help secure PC supplies in areas experiencing YFV outbreaks where massive vaccination campaigns are required.

Flow path system of ultraviolet C irradiation from xenon flash to reduce bacteria survival in platelet products containing a platelet additive solution.

BACKGROUND: Our previous study showed that ultraviolet C (UVC) from xenon (Xe) flash without any photoreactive compounds inactivated bacteria in platelet concentrates (PCs) with less damage to platelets (PLTs) as compared with Xe flash containing ultraviolet A, ultraviolet B, and visible light. Here, we report a UVC irradiation system for PCs under flow conditions consisting of a flow path-irradiation sheet, a peristaltic pump, and a collection bag. STUDY DESIGN AND METHODS: Platelet concentrates containing Ringer's solution (R-PCs) inoculated with bacteria were injected into a flow path sheet using a peristaltic pump, being irradiated with UVC from Xe flash. The quality of the irradiated PCs containing platelet additive solution (PAS-PCs) was assessed based on PC variables, PLT surface markers, and aggregation ability. RESULTS: Streptococcus dysgalactiae (12 tests) and Escherichia coli (11) were all negative on bacterial culture, while Staphylococcus aureus (12) and Klebsiella pneumoniae (14) grew in one and two R-PCs, respectively. Bacillus cereus spores were inactivated in 7 of 12 R-PCs. PC variables became significantly different between irradiated and nonirradiated PAS-PCs. P-selectin, first procaspase-activating compound (PAC-1) binding, and phosphatidylserine increased by irradiation. Aggregability stimulated by adenosine diphosphate, collagen, or thromboxane A2 increased in the irradiated PAS-PCs, while that by thrombin became smaller compared with nonirradiated controls. CONCLUSION: This newly developed system inactivated bacteria including spores in R-PCs. PAS-PCs irradiated by this system retained acceptable in vitro quality and aggregability. Usage of a peristaltic pump instead of agitator during irradiation may enable this system to be directly combined with an apheresis blood cell separator.

The neuroprotective activity of heat-treated human platelet lysate biomaterials manufactured from outdated pathogen-reduced (amotosalen/UVA) platelet concentrates.

BACKGROUND: Effective neurorestorative therapies of neurodegenerative diseases must be developed. There is increasing interest in using human platelet lysates, rich in neurotrophic factors, as novel disease-modifying strategy of neurodegeneration. To ensure virus safety, pathogen reduction treatments should be incorporated in the preparation process of the platelet concentrates used as source material. We therefore investigated whether platelet concentrates (PC) pathogen-inactivated using a licensed photo-inactivation treatment combining photosensitive psoralen (amotosalen) and UVA irradiation (Intercept) can serve as source material to prepare platelet lysates with preserved neuroprotective activity in Parkinson's disease models. METHODS: Intercept treated-PCs were centrifuged, when reaching expiry day (7 days after collection), to remove plasma and platelet additive solution. The platelet pellet was re-suspended and concentrated in phosphate buffer saline, subjected to 3 freeze-thaw cycles (- 80 °C/37 °C) then centrifuged to remove cell debris. The supernatant was recovered and further purified, or not, by heat-treatment as in our previous investigations. The content in proteins and neurotrophic factors was determined and the toxicity and neuroprotective activity of the platelet lysates towards LUHMES cells or primary cortical/hippocampal neurons were assessed using ELISA, flow cytometry, cell viability and cytotoxicity assays and proteins analysis by Western blot. RESULTS: Platelet lysates contained the expected level of total proteins (ca. 7-14 mg/mL) and neurotrophic factors. Virally inactivated and heat-treated platelet lysates did not exert detectable toxic effects on neither Lund human mesencephalic dopaminergic LUHMES cell line nor primary neurons. When used at doses of 5 and 0.5%, they enhanced the expression of tyrosine hydroxylase and neuron-specific enolase in LUHMES cells and did not significantly impact synaptic protein expression in primary neurons, respectively. Furthermore, virally-inactivated platelet lysates tested were found to exert very strong neuroprotection effects on both LUHMES and primary neurons exposed to erastin, an inducer of ferroptosis cell death. CONCLUSION: Outdated Intercept pathogen-reduced platelet concentrates can be used to prepare safe and highly neuroprotective human heat-treated platelet pellet lysates. These data open reassuring perspectives in the possibility to develop an effective biotherapy using virally-inactivated platelet lysates rich in functional neurotrophins for neuroregenerative medicine, and for further bio-industrial development. However, the data should be confirmed in animal models.

Pathogen inactivation with amotosalen plus UVA illumination minimally impacts microRNA expression in platelets during storage under standard blood banking conditions.

BACKGROUND: To reduce the risk of transfusion transmission infection, nucleic acid targeted methods have been developed to inactivate pathogens in PCs. miRNAs have been shown to play an important role in platelet function, and changes in the abundance of specific miRNAs during storage have been observed, as have perturbation effects related to pathogen inactivation (PI) methods. The aim of this work was to investigate the effects of PI on selected miRNAs during storage. STUDY DESIGN AND METHODS: Using a pool and split strategy, 3 identical buffy coat PC units were generated from a pool of 24 whole blood donors. Each unit received a different treatment: 1) Untreated platelet control in platelet additive solution (C-PAS); 2) Amotosalen-UVA-treated platelets in PAS (PI-PAS); and 3) untreated platelets in donor plasma (U-PL). PCs were stored for 7 days under standard blood banking conditions. Standard platelet quality control (QC) parameters and 25 selected miRNAs were analyzed. RESULTS: During the 7-day storage period, differences were found in several QC parameters relating to PI treatment and storage in plasma, but overall the three treatments were comparable. Out of 25 miRNA tested changes in regulation of 5 miRNA in PI-PAS and 3 miRNA U-PL where detected compared to C-PAS. A statistically significant difference was observed in down regulations miR-96-5p on Days 2 and 4, 61.9% and 61.8%, respectively, in the PI-PAS treatment. CONCLUSION: Amotosalen-UVA treatment does not significantly alter the miRNA profile of platelet concentrates generated and stored using standard blood banking conditions.

Clinical impact of amotosalen-ultraviolet A pathogen-inactivated platelets stored for up to 7 days.

BACKGROUND: Universal pathogen inactivation of platelet concentrates (PCs) using amotosalen/ultraviolet A with 7-day storage was implemented in Switzerland in 2011. Routine-use data were analyzed at the University Hospital Basel, Switzerland. STUDY DESIGN: A retrospective two-cohort study of patient and PC characteristics, component usage, patient outcomes, count increments (CIs), and adverse events were analyzed for two consecutive 5-year periods with either 0- to 5-day-old conventional PC (C-PC) (n = 14,181) or 0- to 7-day-old pathogen-inactivated PC (PI-PC) (n = 22,579). RESULTS: In both periods, PCs were issued for transfusion on a "first in, first out" basis. With 7-day PI-PC, wastage was reduced from 8.7% to 1.5%; 16.6% of transfused PI-PCs were more than 5 days old. Transfusion of PI-PC more than 5 days old compared with 5 days old or less did not increase platelet and RBC use on the same or next day as an indirect measure of hemostasis and did not increase transfusion reactions. Mean corrected count increments (CCIs) for PI-PC stored for 5 days or less were 22.6% lower than for C-PC (p < 0.001), and declined with increasing storage duration for both, although the correlation was weak (r2 = 0.005-0.014). Mean number of PCs used per patient and duration of PC support were not different for hematology/oncology, allogeneic and autologous hematopoietic stem cell transplant (HSCT), and general medical/surgical patients, who used the majority (~92.0%) of PI-PCs. Five-year treatment-related mortality in allogeneic HSCT was unchanged in the PI-PC period. CONCLUSIONS: PI-PCs with 7-day storage reduced wastage and did not increase PC or red blood cell utilization or adverse reactions compared with fresh PI-PC or a historical control group, demonstrating preserved efficacy and safety.

Allogeneic major histocompatibility complex antigens are necessary and sufficient for partial tolerance induced by transfusion of pathogen reduced platelets in mice.

BACKGROUND AND OBJECTIVES: Alloimmunization is common following transfusion with platelet-rich plasma (PRP) and can cause complications such as platelet refractoriness or transplant rejection. It has previously been shown that pathogen reduction of PRP with riboflavin and UV light (UV+R) can protect against alloimmunization in mice and induce partial tolerance to subsequent transfusions. MATERIALS AND METHODS: Using B6 H2d congenic mice, this study evaluated the relative contributions of major histocompatibility complex (MHC) antigens and minor antigens to both the alloresponse to PRP transfusion and the partial tolerance induced by UV+R treatment. RESULTS: Both total and MHC-specific alloantibody responses were highest when both MHC and minor antigens were mismatched, with lower alloantibody responses observed with MHC mismatch alone, demonstrating that allogeneic minor antigens can enhance the response to allogeneic MHC. There was a weak, but significant alloantibody response to minor antigens only. UV+R treatment protected against both major and minor antigen alloimmunization. Both allogeneic MHC and minor antigens primed an enhanced cytokine response ex vivo, though this was weaker with minor antigens, and both responses were blocked with UV+R treatment. CONCLUSION: Allogeneic MHC is both necessary and sufficient to induce the partial tolerance associated with UV+R treatment.

Ultraviolet C light efficiently inactivates nonenveloped hepatitis A virus and feline calicivirus in platelet concentrates.

BACKGROUND: Nonenveloped transfusion-transmissible viruses such as hepatitis A virus (HAV) and hepatitis E virus (HEV) are resistant to many of the common virus inactivation procedures for blood products. This study investigated the pathogen inactivation (PI) efficacy of the THERAFLEX UV-Platelets system against two nonenveloped viruses: HAV and feline calicivirus (FCV), in platelet concentrates (PCs). STUDY DESIGN AND METHODS: PCs in additive solution were spiked with high titers of cell culture-derived HAV and FCV, and treated with ultraviolet C at various doses. Pre- and posttreatment samples were taken and the level of viral infectivity determined at each dose. For some samples, large-volume plating was performed to improve the detection limit of the virus assay. RESULTS: THERAFLEX UV-Platelets reduced HAV titers in PCs to the limit of detection, resulting in a virus reduction factor of greater than 4.2 log steps, and reduced FCV infectivity in PCs by 3.0 ± 0.2 log steps. CONCLUSIONS: THERAFLEX UV-Platelets effectively inactivates HAV and FCV in platelet units.

Ultraviolet light-based pathogen inactivation and alloimmunization after platelet transfusion: results from a randomized trial.

BACKGROUND: The current study explored whether pathogen-reduction treatment of platelet components before transfusion would decrease the risk of alloimmunization. STUDY DESIGN AND METHODS: Study participants were patients with hematologic cancer who were included in two parallel, randomized clinical trials testing pathogen-reduction treatment versus conventional platelets using the Mirasol or Intercept pathogen-reduction systems. Patients who had a baseline, pretransfusion sample and a follow-up, posttransfusion sample were included in the study (n = 179 patients in each study arm). Human leukocyte antigen antibody levels were determined using a commercial multianalyte, bead-based assay. RESULTS: The rate of human leukocyte antigen Class I alloimmunization at the clinical sites in recipients of conventional platelets was low at the highest assay cutoff (range, 1.2%-5.9%). Consistent with prior studies, human leukocyte antigen antibodies were first detected from 3 to 35 days after transfusion. There were no statistically significant differences between alloimmunization rates in patients who received pathogen-reduction treatment versus conventional platelet transfusions. Although he difference was not statistically significant, the effect size for protection from alloimmunization was greatest for high-level human leukocyte antigen Class I antibodies (approximately threefold) in the Intercept-treated patients compared with those who received conventional platelets. In the Mirasol study, only two patients and one patient in the control group developed medium-level or high-level antibodies, respectively, so it was impossible to determine an effect size for potential protection. CONCLUSIONS: The current study was not sufficiently powered to determine whether pathogen-reduction treatment provides protection from human leukocyte antigen alloimmunization in platelet transfusion recipients. The data presented will be useful in the design of future trials and endpoints powered to detect a protective effect.