Platelet Concentrates Storage Research Articles

Proliferation of psychrotrophic bacteria in cold-stored platelet concentrates.

Platelet concentrates (PC) are stored at 20-24°C to maintain platelet functionality, which may promote growth of contaminant bacteria. Alternatively, cold storage of PC limits bacterial growth; however, data related to proliferation of psychotrophic species in cold-stored PC (CSP) are scarce, which is addressed in this study. Eight laboratories participated in this study with a pool/split approach. Two split PC units were spiked with ~25 colony forming units (CFU)/PC of Staphylococcus aureus, Klebsiella pneumoniae, Serratia liquefaciens, Pseudomonas fluorescens and Listeria monocytogenes. One unit was stored under agitation at 20-24°C/7 days while the second was stored at 1-6°C/no agitation for 21 days. PC were sampled periodically to determine bacterial loads. Five laboratories repeated the study with PC inoculated with lyophilized inocula (~30 CFU/mL) of S. aureus and K. pneumoniae. All species proliferated in PC stored at 20-24°C, reaching concentrations of ≤109 CFU/mL by day 7. Psychrotrophic P. fluorescens and S. liquefaciens proliferated in CSP to ~106 CFU/mL and ~105 CFU/mL on days 10 and 17 of storage, respectively, followed by L. monocytogenes, which reached ~102 CFU/mL on day 21. S. aureus and K. pneumoniae did not grow in CSP. Psychrotrophic bacteria, which are relatively rare contaminants in PC, proliferated in CSP, with P. fluorescens reaching clinically significant levels (≥105 CFU/mL) before day 14 of storage. Cold storage reduces bacterial risk of PC to levels comparable with RBC units. Safety of CSP could be further improved by implementing bacterial detection systems or pathogen reduction technologies if storage is beyond 10 days.

In platelet single donor apheresis, platelet factor 4 levels correlated with donor's age and decreased during storage.

The human population is ageing worldwide. The World Health Organization estimated that the world's population of people aged60years and older will increase to at least 30%, coinciding with a growing frequency of cognitive and cardiovascular disease. Recently, in preclinical studies platelet Factor 4 (PF4) was presented as a pro-cognitive factor. This molecule is released by platelets in the circulation and could be present in blood products destined for transfusion. We wondered if PF4 levels are correlated to the age of the blood donor or to the storage time of platelet concentrates (PCs) intended for transfusion? We observed higher levels of PF4 in PCs from elderly donors compared to younger donors, while PC storage time did not determine PF4 levels expression.

A tight interplay between platelet activation and mitochondrial DNA release promotes platelet storage lesion in platelet concentrates.

Platelet storage lesion (PSL) adversely affects the quality of platelet concentrates (PCs). Platelets are prone to activation during storage. Moreover, elevated free mitochondrial DNA (mtDNA) levels in PCs are associated with a higher risk of adverse transfusion reactions. Therefore, we aimed to evaluate the correlation between platelet activation markers and mtDNA release during PC storage. Six PCs prepared by the platelet-rich plasma method were assessed for free mtDNA copy number using quantitative real-time PCR and CD62P (P-selectin) expression by flow cytometry on days 0 (PC collection day), 3, 5 and 7 of storage. Lactate dehydrogenase (LDH) activity, pH, platelet count, mean platelet volume (MPV) and platelet distribution width (PDW) were measured as well. The correlation between free mtDNA and other PSL parameters, and the correlation between all parameters, was determined. Significant increases in free mtDNA, MPV and PDW, and a significant decrease in platelet count and pH were observed. CD62P expression and LDH activity elevated significantly, particularly on storage days 5-7 and 0-3, respectively. Moreover, a moderate positive correlation (r = 0.61) was observed between free mtDNA and CD62P expression. The r values between free mtDNA and LDH, pH, platelet count, MPV and PDW were 0.81, -0.72, -0.49, 0.81 and 0.77, respectively. The interplay between platelet activation and mtDNA release in promoting PSL in PCs may serve as a promising target for future research on applying additive solutions and evaluating the quality of PCs to improve transfusion and clinical outcomes.

Apoptotic Pathway Inhibition in Cold-Stored Platelet Concentrates Facilitates Retained Ability to Form Thrombi

Introduction: Platelet concentrates (PCs) are used to treat or prevent bleeding in patients with impaired platelet function or after injury. However, the standard storage of PCs at room-temperature (RT) increases the risk of bacterial contamination and is associated with reduced platelet functionality due to storage lesions. Our group reported that cold-stored PCs have better functionality compared to RT-stored PCs but reduced in vivo circulation time due to cold-induced apoptosis. In order to investigate cold-stored PCs hemostatic functionality with and without apoptosis inhibition, we established an ex vivo model simulating physiological conditions of blood flow to evaluate the contribution of cold-stored PCs in thrombus formation. Methods: Apheresis-derived PCs were stored for 1, 4, 7 and 10 days under agitation at 4°C or RT either with or without the apoptosis inhibitor G04 (RhoA GTPase inhibitor). Next, PCs were stained with CD41 antibody, recalcified and incubated with TRAP6 (thrombin receptor-activating peptide 6) to initiate thrombus formation. Platelet-depleted whole blood samples from healthy donors were spiked-in with PCs and administered to the microfluidic channels of the ex vivo system (Bioflux), under physiological shear pressure. After five minutes of perfusion the resulting thrombi were imaged and analyzed. Ability to form thrombi was further assessed by thromboelastography and impedance aggregometry. Results: Platelets from 24h storage PCs showed stable thrombus formation upon activation with TRAP6. Unstimulated platelets did not form any thrombi during the entire period of perfusion. To evaluate the effect of cold storage on platelet functions we analyzed the extent of thrombus formation of PCs stored at RT and 4°C upon CD41 staining. Our results indicate that cold-stored platelets form larger clots under flow conditions and maintain higher functionality compared to PCs stored at RT. Moreover, incubation of cold-stored PCs with the apoptosis inhibitor G04 improved ex vivo thrombus formation compared to PCs stored without the inhibitor, without inducing any adverse effects (Figure 1). Conclusion: The results indicate that our ex vivo assay, which simulates PC transfusion in thrombocytopenic patients, is suitable to test the hemostatic functions of PCs under physiological flow conditions. We demonstrated that PCs stored at 4°C show better thrombus formation ability upon stimulation by agonists compared to RT-stored PCs and that the ability to form clots under shear stress is still preserved after incubation with the apoptosis inhibitor. To conclude, cold-storage of PCs in combination with apoptosis inhibition is a promising stragedy to reduce the risk of bacterial contamination and prolong in vivo survival time while preserving platelet's hemostatic functionality.

Biochemical and functional characteristics of stored (double-dose) buffy-coat platelet concentrates treated with amotosalen and a prototype UVA light-emitting diode illuminator.

Pathogen reduction of platelet concentrates (PCs) using amotosalen and broad-spectrum UVA illumination contributes to the safety of platelet transfusion by reducing the risk of transfusion-transmitted infections. We evaluated the in vitro quality of stored buffy-coat (BC) PCs treated with amotosalen and a prototype light-emitting diode (LED) illuminator. Double-dose BC-PCs collected into PAS-III/plasma or SSP+ /plasma (55/45%) were treated with amotosalen in combination with either conventional UVA lamps (INT100 Illuminator 320-400 nm) or LED illuminators at 350 nm. Platelet quality and function were evaluated over 7 days. Platelet counts were conserved during storage in all groups, as was platelet swirling without appearance of macroscopic aggregates. Integrin αIIbβ3 and glycoprotein (GP) VI expression remained stable, whereas GPIbα and GPV declined similarly in all groups. UV lamp- and LED-treated PCs displayed similar glucose consumption, lactate generation, and pH variation. Comparable spontaneous and residual P-selectin and phosphatidylserine exposure, activated αIIbβ3 exposure, mitochondrial membrane potential, lactate dehydrogenase release, and adhesive properties under flow conditions were observed during storage. The use of SSP+ /plasma compared with PAS-III/plasma better preserved most of these parameters, especially during late storage, irrespective of the type of illuminator. Replacing the UVA lamp for photochemical treatment by LED illuminators had no impact on platelet metabolism, spontaneous activation, apoptosis or viability, or on the in vitro function of BC-PCs stored for 7 days in SSP+ or PAS-III/plasma. These findings support improved procedures for the pathogen reduction and storage of PCs, to ensure transfusion safety and retention of platelet functional properties.

Quantitative increases of extracellular vesicles in prolonged cold storage of platelets increases the potential to enhance fibrin clot formation.

Platelet derived extracellular vesicles (EVs) display a pro-coagulant phenotype and are generated throughout platelet concentrate (PC) storage. Cold storage (CS) of PCs is thought to provide a superior haemostatic advantage over room temperature (RT) storage and could prolong the storage time. However, the effect of storage conditions on EV generation and PC function is unknown. We investigated EV production under CS and RT conditions and assessed whether these EVs exhibited a more pro-coagulant phenotype in model experiments. Buffy-coat-derived PCs in a platelet additive solution (PAS) to plasma ratio of approximately 65:35 were stored at RT (22 ± 2°C) or CS (4 ± 2°C) for a prolonged storage duration of 20 days. Impedance aggregometry assessed platelet function. EVs were isolated throughout storage and quantified using nanoparticle tracking analysis. EVs were applied to a coagulation assay to assess the impact on fibrin clot formation and lysis. CS produced significantly larger EVs from day 4 onwards. EV concentration was significantly increased in CS compared to RT from day 15. EVs, regardless of storage, significantly reduced time to clot formation and maximum optical density measured compared to the no EV control. Clot formation was proportionate to the number of EV applied but was not statistically different across storage conditions when corrected for EV number. EVs in CS and RT units showed similar clot formation capacity. However, the higher number of larger EVs generated in CS compared to RT suggests PC units derived from CS conditions may overall exhibit a haemostatically superior capacity compared to RT storage.

Resveratrol reduces platelet storage lesion by preventing free mitochondrial DNA (mtDNA) accumulation in platelet concentrates during storage.

Platelet activation and mitochondrial damage are among the crucial events leading to the quality reduction of platelet concentrates (PCs) during preparation and storage, called platelet storage lesion. Platelet activation results in the clearance of transfused platelets. Oxidative stress and platelet activation trigger mitochondrial DNA (mtDNA) release into the extracellular milieu which is associated with adverse transfusion reactions. Therefore, we aimed to investigate the effects of resveratrol, an antioxidant polyphenol, on platelet activation markers and mtDNA release. Ten PCs were divided equally into two bags each, one of them was allocated to the control group (n = 10) and another to the case group (resveratrol-treated, n = 10). Free mtDNA level and CD62P (P-selectin) expression level were measured by absolute quantification Real-Time PCR, and flow cytometry on days 0 (the receiving day), 3, 5, and 7 of storage respectively. Moreover, Lactate dehydrogenase (LDH) enzyme activity, pH, platelet count, mean platelet volume (MPV), and platelet distribution width (PDW) were assessed as well. Treatment of PCs with resveratrol can significantly decrease mtDNA release during storage compared to the control. In addition, platelet activation was significantly mitigated. We also observed significantly lower MPV, PDW, and LDH activity in resveratrol-treated PCs compared to the control group on days 3, 5, and 7. Furthermore, resveratrol maintained the pH of PCs on day 7.Resveratrol diminished free mtDNA and maintained biochemical parameters in PCs, possibly by reducing platelet activation. Therefore, resveratrol might be a possible additive solution for improving the quality of stored PCs.

Proof of concept for detection of staphylococcal enterotoxins in platelet concentrates as a novel safety mitigation strategy.

Staphylococcus aureus is a predominant contaminant of platelet concentrates (PCs) that can evade detection during screening with culture methods. Importantly, S. aureus produces staphylococcal enterotoxins (SEs) during PC storage, which are linked to slow growth and enhanced biofilm formation. This study investigated timing of SE production during PC storage and feasibility of SE detection as a PC safety strategy. Genomic and transcriptomic data of transfusion-relevant S. aureus PS/BAC/169/17/W, PS/BAC/317/16/W, CI/BAC/25/13/W and CBS2016-05 were used to determine the presence and differential expression of exotoxin genes in PCs. Trypticase soy broth (TSB) and PCs were inoculated with 1.0E+06 cfu/mL of S. aureus PS/BAC/169/17/W and CBS2016-05. Expression of SEs at different growth phases was confirmed with Western blotting. PCs were inoculated with 30 cfu/unit of the same strains, and SE detection during PC storage was optimized with a sandwich dot-ELISA assay. S. aureus genomes contain multiple exotoxin genes including those encoding for SEs. Transcriptome data revealed significant upregulation (0.5-6.7-fold, p < 0.05) of SE genes in PCs versus TSB. Western blots demonstrated SE production at all growth phases. Notably, dot-ELISA detected clinically relevant concentrations of SEs (~0.2 μg/mL) at 32 h of PC storage when S. aureus PS/BAC/169/17/W and CBS2016-05 counts were 1.8E+04 and 1.4E+04 cfu/mL, respectively. Genomic analyses revealed that staphylococcal exotoxins are widely distributed and highly conserved among transfusion-relevant S. aureus isolates. Furthermore, SEs are significantly upregulated in PCs and detected at 30 h of PC storage. Therefore, bacterial toxin detection could supplement mitigation strategies to enhance PC safety.

Bioactive lipids as biomarkers of adverse reactions associated with apheresis platelet concentrate transfusion.

Platelet concentrate (PC) transfusion seeks to provide haemostasis in patients presenting severe central thrombocytopenia or severe bleeding. PCs may induce adverse reactions (AR) that can occasionally be severe (SAR). PCs contain active biomolecules such as cytokines and lipid mediators. The processing and storage of PCs creates so-called structural and biochemical storage lesions that accumulate when blood products reach their shelf life. We sought to investigate lipid mediators as bioactive molecules of interest during storage and review associations with adverse reactions post-transfusion. To facilitate understanding, we focused on single donor apheresis (SDA) PCs with approximately 31.8% of PCs being delivered in our setting. Indeed, pooled PCs are the most widely transfused products, but the study of a single donor lipid mediator is easier to interpret. We are investigating key lipid mediators involved in AR. Adverse reactions were closely monitored in accordance with current national and regional haemovigilance protocols. Residual PCs were analysed post-transfusion in a series of observations, both with and without severe reactions in recipients. A decrease in the lysophosphatidylcholine species to produce the lysophosphatidic acid species has been observed during storage and in the case of AR. Lysophosphatidic acid increased with primarily platelet-inhibitor lipids. Anti-inflammatory platelet-induced inhibition lipids were weakly expressed in cases of severe adverse reactions. We therefore propose that a decrease in lysophosphatidylcholine and an increase in lysophosphatidic acid can prospectively predict serious adverse transfusion reactions.

Clinically Expired Platelet Concentrates as a Source of Extracellular Vesicles for Targeted Anti-Cancer Drug Delivery.

The short shelf life of platelet concentrates (PC) of up to 5-7 days leads to higher wastage due to expiry. To address this massive financial burden on the healthcare system, alternative applications for expired PC have emerged in recent years. Engineered nanocarriers functionalized with platelet membranes have shown excellent targeting abilities for tumor cells owing to their platelet membrane proteins. Nevertheless, synthetic drug delivery strategies have significant drawbacks that platelet-derived extracellular vesicles (pEV) can overcome. We investigated, for the first time, the use of pEV as a carrier of the anti-breast cancer drug paclitaxel, considering it as an appealing alternative to improve the therapeutic potential of expired PC. The pEV released during PC storage showed a typical EV size distribution profile (100-300 nm) with a cup-shaped morphology. Paclitaxel-loaded pEV showed significant anti-cancer effects in vitro, as demonstrated by their anti-migratory (>30%), anti-angiogenic (>30%), and anti-invasive (>70%) properties in distinct cells found in the breast tumor microenvironment. We provide evidence for a novel application for expired PC by suggesting that the field of tumor treatment research may be broadened by the use of natural carriers.

Resveratrol; a Double-Edged Sword Antioxidant Agent for Preserving Platelet Cell Functions During Storage; Molecular Insights.

In the current study we have aimed to find the effects of Resveratrol treatment on platelet concentrates (PCs) at the dose dependent manner. We have also attempted to find the molecular mechanism of the effects. The PCs, have received from Iranian blood transfusion organization (IBTO). Totally 10 PCs were studied. The PCs divided into 4 groups including untreated (control) and treated by different dose of Resveratrol; 10, 30 and 50 µM. Platelet aggregation and total reactive oxygen species (ROS) levels were evaluated at day 3 of PCs storage. In silico analysis was carried out to find out the potential involved mechanisms. The aggregation against collagen has fallen dramatically in all studied groups but at the same time, aggregation was significantly higher in the control versus treated groups (p<0.05). The inhibitory effect was dose dependent. The aggregation against Ristocetin did not significantly affect by Resveratrol treatment. The mean of total ROS significantly increased in all studied groups except those PCs treated with 10 µM of Resveratrol (P=0.9). The ROS level significantly increased with increasing Resveratrol concentration even more than control group (slope=11.6, P=0.0034). Resveratrol could potently interact with more than 15 different genes which, 10 of them enrolled in cellular regulation of the oxidative stress. Our findings indicated that the Resveratrol affect the platelet aggregation at the dose dependent manner. Moreover, we have also found that the Resveratrol play as double-edged sword in the controlling oxidative state of the cells. Therefore, Using the optimal dose of Resveratrol is the great of importance.

L-carnitine modulates free mitochondrial DNA DAMPs and platelet storage lesions during storage of platelet concentrates.

Platelet storage lesions may occur in Platelet concentrates (PCs) storage time, reducing PCs' quality. Mitochondrial damage causes mitochondrial DNA (mtDNA) to be released into the extracellular space. In this study, we evaluated the effect of L-carnitine (LC) as an antioxidant on free mtDNA DAMPs release in PCs during storage. Ten PCs prepared by the PRP method were studied. The copy numbers of free mtDNA, total reactive oxygen species (ROS), lactate dehydrogenase (LDH) enzyme activity, pH, and platelet counts were measured on days 0, 3, 5, and 7 of PCs storage in LC-treated and untreated platelets. LDH activity was significantly lower than the control group during 7 days of PCs storage (p = 0.041). Also, ROS production decreased in LC-treated PCs compared to the control group during storage (p = 0.026), and the difference mean of ROS between the two groups was significant on day 3, 5, and 7 (Pday3 = 0.02, Pday5 = 0.0001, Pday7 = 0.031). Moreover, LC decreased the copy numbers of free mtDNA during 7 days of storage (p = 0.021), and the difference mean of the copy numbers of free mtDNA in LC-treated PCs compared to the control group was significant on day 5 and 7 (Pday5 = 0.041، Pday7 = 0.022). It seems that LC can maintain the metabolism and antioxidant capacity of PCs and thus can reduce mitochondrial damage and mtDNA release; consequently, it can decrease DAMPs in PCs. Therefore, it may be possible to use this substance as a platelet additive solution in the future.

Contamination of platelet concentrates with Staphylococcus aureus induces significant modulations in platelet functionality

Platelet concentrates (PCs) contaminated with Staphylococcus aureus can escape detection during PC screening, causing septic transfusion reactions. This study aimed to determine the impact of S. aureus contamination on platelet metabolism and functionality during PC storage. Targeted metabolomics (N=3) was performed on non-spiked PCs and PCs inoculated with 10-20 colony-forming units (CFU)/bag of S. aureus. Metabolites were quantified at 0, 48 and 144 h using high-performance mass spectrometry (MS). Additionally, PCs spiked with approximately 20 CFU/bag of S. aureus were sampled every 24 h for up to 144 h to evaluate platelet functionality using flow cytometry (N=2). Eight metabolites had significantly different levels in spiked PCs (log2 fold-change ≤ or ≥±1) versus non-spiked units at 48 and 144 h. Xanthine, uridine, serine, glutamine and threonine were increased, whereas orotic acid, dihydroorotic acid and aspartic acid were decreased. Flow cytometry showed a significant decrease in expression of GPIIb while P-selectin expression was significantly increased in spiked PCs after 72 h of storage when S. aureus concentration was ≥10E+08 CFU/ml. Additionally, phosphatidylserine exposure was significantly increased after 48 h of PC storage, when S. aureus had reached a concentration of 2E+06. Contamination with S. aureus exacerbates platelet storage lesions in contaminated PCs but only when the bacterium has reached clinically significant levels.

Utilization of Expired Platelet Concentrate for Production of Human Platelet Lysate as a Medium for T47D Cell Propagation

Background: Platelet concentrate (PC) has a short shelf life (5 days). Expired PC cannot be used for clinical purposes. PC is used for human platelet lysate (HPL) production, which was found to be more effective than FBS at increasing T47D cell proliferation. HPL production using expired PC has not been reported. This study aimed to investigate whether the use of HPL produced from expired PC (storage duration &gt;5 days) can increase the proliferation of T47D cells in vitro.Materials and methods: Expired PC samples with a shelf life of 7 and 11 days were used to produce HPL via freeze/thaw method. pH, total protein content, glucose and albumin levels were measured. The 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay was used to measure proliferation rate and doubling time of HPL-treated T47D cells.Results: After HPL production, the glucose level was influenced by the pH (p=0.003), and albumin level was influenced by total protein content (p=0.030). HPL stored for 7 and 11 days increased cell proliferation rate by 1.41 and 1.80 times higher than 10% FBS, respectively. HPL produced from expired PC did not cause morphological abnormality of the cells. In this study, the glucose levels affected cell proliferation (p=0.030). High glucose levels inhibited T47D cell proliferation.Conclusion: Expired PC can be used as a potential material for HPL production, since HPL produced from expired PC increases cell proliferation rate and shortens cell doubling time.Keywords: cell proliferation, human platelet lysate, platelet concentrate, thrombocyte, T47D

Platelet Refractory Cases after Transfusion of Fresh and Stored Platelet in all Children

Introduction: Acute leukemia is the most common form of cancer in children, platelet transfusion is needed for the chemotherapy of acute lymphoblastic leukaemia children. Platelets are transfused for therapeutic and prophylactic purposes. Transfusion of stored platelet concentrates (up to 5 days) has been demonstrated to be as effective as transfusion of fresh platelet concentrates (upto 24 hours). Platelet refractory cases were also observed in this study that was detected by Corrected Count Increment (CCI) and Percent Platelet Recovery (PPI) at 1hour and 24 hours. Aim of the study: The aim of this study was to observe the platelet refractory cases after transfusion of fresh and stored platelet in acute lymphoblastic leukemia (ALL) in a tertiary care hospital, Dhaka. Methods: A cross-sectional study was conducted in the Department of Clinical Pathology, Paediatric Haematolo- Oncology and transfusion Medicine BSMMU, Department of Haematology and Paediatric Haemato-Oncology in Dhaka Medical College Hospital from March 2010 to February 2011 where 81 children diagnosed with acute lymphoblastic leukaemia were taken as the study population. Non probability purposive sampling was used by fulfilling the inclusion criteria. Ethical consideration was taken by the BSMMU ethical review committee. A data sheet with two parts (Part A and Part B) was designed with a view to collect data from the patients to be enrolled in the study. Data were analyzed using the SPSS version 25.0. Results: Out of 81 in 47 children with acute lymphoblastic leukemia (ALL), fresh platelet concentrates (FPC) (upto 24 hours) or day-0 platelets were transfused. In 34 children, stored platelet concentrates (SPC) (days 1–5) were transfused. In 27 cases, platelet concentrates were transfused both fresh and stored. In FPC and SPC, the corrected count increment at one hour (CCI 1h) was 20.5×109/L and 18.9×109/L respectively. FPC and SPC values for the mean corrected count increment at 24 hours (CCI 24h) were 15.5×109/L and 13.8×109/L respectively. Platelet refractory cases were 15(18.5%) out of 81 cases transfused. Platelet refractory cases were 7(15%) out of 47 cases transfused with FPC and 8(23%) out of 34 cases transfused with stored platelet concentrate (SPC). There were no significant differences (P&lt;0.05) between FPC and SPC according to platelet refractoriness. Conclusion: In vivo quality of freshly generated platelet (upto 24 hours) versus stored platelet concentrates (up to 5 days) were determined after 1 hour and 24 hours. 1-5 day’s stored PRP-PCs are as effective as fresh PRP-PCs for sufficient platelet increment.

Expression of biofilm-associated genes in Staphylococcus aureus during storage of platelet concentrates

Background and objectivesIn transfusion medicine, the safety of platelet concentrates (PCs) is a major concern on account of contamination, mostly with Staphylococcus species. One of the most common contaminants is Staphylococcus aureus, which forms bacterial biofilms in PCs, posing a safety risk for transfusion patients. In this study, we investigate the contributions to biofilm formation of eno, ebps, and fib genes encoding surface proteins and of genes from the ica operon (icaA and icaD) encoding polysaccharide intercellular adhesin (PIA), along with their expression in bacteria grown in glucose-supplemented trypticase soy broth (TSBg) and PCs. Materials and methodsTwo strains of S. aureus (2039 and 2110) captured during routine PC screening were tested for biofilm formation in TSBg and under PC storage conditions, with mRNA collected at five time points and analyzed to determine expression of eno, ebps, fib, icaA, and icaD and their contributions to biofilm formation in both media. ResultsIn TSBg, S. aureus strain 2039 formed weak biofilms while 2110 formed strong. biofilms; however, in PCs, both strains formed strong biofilms. During biofilm formation, expression levels of icaA and icaD in both strains were generally significantly higher in TSBg than PCs. In contrast, expression of eno, ebps, and fib genes tended to be significantly higher under PC storage conditions. ConclusionThis study demonstrated that expression of genes involved in biofilm formation can be affected by growth media. Further investigation is needed to understand biofilm formation in the PC milieu and enhance transfusion safety.

Abstract 158: Bioactive Lipids As Biomarkers Of Severe Adverse Reaction Associated With Apheresis Platelet Concentrate Transfusion

Blood transfusion is a life-saving procedure in which whole blood, or blood components are provided to a patient. Platelet concentrates (PC) may sometimes induce adverse reactions, that are occasionally severe. PC comprise active biomolecules such as cytokines and lipid mediators. Platelet storage lesions are structural and biochemical changes in PC and vary in collection and processing conditions. We investigated lipid mediators as bioactive molecules along storage conditions and in adverse reactions after transfusion. The PC left over were analysed after transfusion, classified with/out severe reaction. Our data presented a decrease of lysophosphatidylcholine species to produce lysophosphatidic acid species. This balance could lead and be correlated with complication symptoms, as other lipids. Lysophosphatidic acid increased with primarily platelet-inhibitor-lipids when single donor apheresis platelet upon storage. Anti-inflammatory and platelet-induced-inhibition lipids were weakly expressed in case of severe adverse reaction. Thus, a decrease in lysophosphatidylcholine and an increase in lysophosphatidic acid could be reliable predictors of serious adverse transfusion reactions transfusion. These data highlighted the importance of PC manufacturing and storage processing, not only to maximize clinical efficacy but also to minimize adverse reactions.

In vitro characteristics of cryopreserved platelet concentrates reconstituted by fresh frozen or lyophilized plasma.

Six percent dimethyl sulfoxide (DMSO) cryopreservation of platelet concentrates (PCs) allow longer storage of PCs but require time-consuming post-thaw washing. An alternative process based on removing supernatant before freezing has been implemented in several centres worldwide. We assessed the in vitro characteristics of cryopreserved PCs (CPPs) prepared according to this latest process using either French lyophilized plasma (FLyP) or fresh frozen plasma (FFP) for reconstitution. FLyP provides additional benefits to the process due to its logistical constraints and quick availability. Apheresis PCs (n=16) and buffy coat PCs (n=16) were cryopreserved in 6% DMSO. After storage at -80°C, PCs were thawed and reconstituted with FFP or FLyP. Volume, residual leukocytes, total platelet counts (TPCs), post-thaw recovery, biochemical parameters, and DMSO concentration were assessed. Platelet functions were analysed by swirling index, viscoelastometric assay and CD62P quantification. After reconstitution, TPC was above 2.1011/CPs; recovery was 78±14% with no significant difference between FFP and FLyP. Glucose and lactate levels were not different between plasmas, whereas FLyP-CPPs exhibited a significant increase in LDH and significantly lower pH. Residual DMSO was 8±4G/L. Functional analysis revealed significant differences between FFP and FLyP-CPPs, with lower clot firmness and increased clot initiation. Activation of platelets was not higher in FLyP-CPPs. Preparing CPPs according to this "new" process fulfilled the French legal criteria regardless of the type of plasma. Differences highlighted between FFP-CPPs and FLyP-CPPs were unlikely to be of clinical relevance.

A Comparative Study between Fresh and Stored Platelet Concentrate after Transfusion in same ALL patients in a Tertiary Hospital

Background: Platelet transfusion is needed for the treatment of ALL children. Researchers have shown that transfusion of stored platelet concentrates (up to 5days) is as effective as fresh platelet concentrates (24 hours). Objectives: The present study was conducted to access the comparison between fresh and stored platelet concentrate after transfusion in same all patients in a tertiary hospital. Materials and methods: The study was a cross-sectional descriptive study which was conducted in department of Clinical Pathology, Paediatric Haemato-Oncology and transfusion Medicine BSMMU, and Department of Haematology and Paediatric Haemato-Oncology in Dhaka Medical College Hospital. Over a period of March 2010 to February 2011.The sample size was 81. A data sheet with two parts (Part A and Part B) was designed with a view to collect data from the patient to be enrolled in the study .The data were analyzed using the SPSS version 25.0. Results: Out of 81 patients fresh platelet concentrates (FPC) or day-0 platelets were transfused in 47 acute Lymphoblastic leukaemia (ALL) children. Stored platelet concentrates (day 1-5) were transfused in 34 children. In 27cases both fresh and stored platelet concentrates were transfused. Corrected count increment at 1 hour (CCI 1h) in FPC and SPC were 20.5 and 18.9 respectively. Mean corrected count increment at 24 hour (CCI 24h) FPC and SPC were 15.5 and 13.8 respectively. There were no significant differences between FPC and SPC P&gt;0.05. Conclusions: It was observed that 1-5 day's stored platelet-rich-plasma platelet concentrate (PRP-PC) was as effective as fresh PC to obtain acceptable platelet increment in childhood acute lymphoblastic leukemia. Furthermore, corrected count increment and percentage platelet recovery are important tools to evaluate platelet refractoriness.

Platelet transfusion practice and related transfusion reactions in a large teaching hospital.

Platelet transfusion practice varies widely since many aspects of platelet concentrate (PC) use have not been definitively determined. The objectives of this retrospective study were to present platelet transfusion practice and evaluate PC and patient characteristics, as well as their association with transfusion reaction (TR) rate. Platelet transfusions over a 5-year period were analysed regarding PC characteristics (the ABO and RhD compatibility, product type, and storage duration), patient characteristics (most responsible diagnosis, age, and gender), and TR type. A total of 46,351 PCs were transfused: 76.4% whole blood-derived (WBD) and 23.6% single donor apheresis (SDA). Three thousand seven hundred seventy-six patients received platelet transfusions: 24.7% paediatric and 75.3% adult patients, 79.6% outpatients and 20.4% inpatients. As much as 63.1% of all transfused PCs were fresh (stored for≤3 days), 98.0% ABO-identical, and 87.3% of all PCs given to RhD- patients were RhD-. PCs were mainly transfused to haemato-oncology (76.8%) and cardiovascular surgery patients (6.5%). Overall, 84 (0.18%) TRs were reported, with allergic TRs (ATRs) being the most common. Although PC ABO compatibility and storage duration, as well as patient age and gender, showed differences in TR rate, only the use of PCs in platelet additive solution (PAS) showed a statistically significant reduction of TRs (P<0.001). Transfusion practice at the University Hospital Centre Zagreb resulted in almost all patients receiving ABO and RhD identical PCs, and most of them were fresh PCs. The most important factor affecting the incidence of TRs was platelet storage solution. The use of PAS effectively reduced the rate of TRs, particularly allergic TRs.