[The Optimal Storage Condition and Storage Time of Umbilical Cord Blood from Collection to Preparation].

OBJECTIVE: To explore the optimal storage condition and time of umbilical cord blood from collection to preparation. METHODS: Collect cord blood samples from 30 healthy newborns, with each new born's umbilical cord blood was divided into two parts on average. One part was stored in cold storage (4 ℃) and the other was stored at room temperature (20-24 ℃). Samples were taken at 24, 36, 48, 60 and 72 h, respectively, total nucleated cells (TNC) count and TNC viability was analyzed. Flow cytometry was used to detect the ratio of viable CD34+ cells to viable CD45+ cells and viability of CD34+ cells, and colony-forming unit-granulocyte-macrophage (CFU-GM) count was performed by hematopoietic progenitor cell colony culture. The change trend of each index over time was observed, and the differences in each index was compared between cold storage and room temperature storage under the same storage time. RESULTS: The TNC count (r 4 ℃=-0.9588， r 20-24 ℃=-0.9790), TNC viability (r 4 ℃=-0.9941， r 20-24 ℃=-0.9970), CD34+ cells viability (r 4 ℃=-0.9932， r 20-24 ℃=-0.9828) of cord blood stored in cold storage (4 ℃) and room temperature storage (20-24 ℃) showed a consistent downward trend with the prolongation of storage time. The percentage of viable CD34+ cells (r 4 ℃=0.9169， r 20-24 ℃=0.7470) and CFU-GM count (r 4 ℃=-0.2537， r 20-24 ℃=-0.8098) did not show consistent trends. When the storage time was the same, the TNC count, TNC viability, CD34+ cells viability and CFU-GM count of cord blood stored in cold storage were higher than those stored at room temperature. Under the same storage time (24, 36, 48, 60 or 72 h), TNC viability in room temperature storage was significantly lower than that in cold storage (P <0.001), but TNC count, percentage of viable CD34+ cells and CFU-GM count were not significantly different between room temperature storage and cold storage. When stored at room temperature for 24 h and 36 h, the viability of CD34+ cells was significantly lower than that in cold storage (P <0.001, P <0.01), when the storage time for 48, 60 and 72 h, there was no significant difference in the CD34+ cells viability between room temperature storage and cold storage. CONCLUSION: It is recommended that cord blood be stored in cold storage (4 ℃) from collection to preparation, and processed as soon as possible.

A perspective on exogenous redox regulation mediated by transfused RBCs subject to the storage lesion.

Granted with a potent ability to interact with and tolerate oxidative stressors, RBCs scavenge most reactive oxygen and nitrogen species (RONS) generated in circulation. This essential non-canonical function, however, renders RBCs susceptible to damage when vascular RONS are generated in excess, making vascular redox imbalance a common etiology of anemia, and thus a common indication for transfusion. This accentuates the relevance of impairments in redox metabolism during hypothermic storage, as the exposure to chronic oxidative stressors upon transfusion could be exceedingly deleterious to stored RBCs. Herein, we review the prominent mechanisms of the hypothermic storage lesion that alter the ability of RBCs to scavenge exogenous RONS as well as the associated clinical relevance.

Drawing and storing blood using a 3D printed bottle cap and a disinfected 500 mL drinking bottle: A proof-of-concept study.

BACKGROUND: Today, with wars raging in Ukraine and the Middle East, the demand for blood is high. Despite this, few companies produce the necessary equipment to draw, store, and transfuse whole blood. This study evaluated the safety and performance of a 3D printed bottle cap in conjunction with a water bottle and some available consumables to draw and store fresh whole blood. STUDY DESIGN AND METHODS: Bags of saline, and freshly donated whole blood, was transferred to the water bottle through a 3D printed bottle cap and stored for 72 h. An identical setup, transferring saline to a Terumo blood collection bag was used as control. Performance and safety were evaluated by calculating infusion rate and observing for backflow, respectively. The blood was also tested for hemolysis and bacterial growth at four sampling points. RESULTS: The cap-and-bottle setup was faster than control in terms of flow rate when transferring saline (1.53 vs. 1.81 mL/s, p < .001), and non-inferior to saline control when transfusing blood (1.53 vs. 1.49 mL/s, p = .641). We did not observe any risks of causing the donor iatrogenic harm, and there was no evidence of increased hemolysis. However, there were traces of bacterial contamination in three of six bottles. CONCLUSION: This study indicates that drawing blood is both feasible and safe, utilizing a 3D printed cap and bottle setup. Flow rate was faster than control, and mechanical properties of the blood were not affected. We were unable to determine the source of bacterial contamination in the blood.

A 3-step method for preparing cryopreserved samples of apheresis products for post-thaw analysis yields a higher percentage of viable cells.

BACKGROUND: Standard flow cytometry protocols for CD34+ cell enumeration designed for fresh samples are not appropriate for cryopreserved products. Special protocols have been developed to remove the cryoprotectant by quickly washing a freshly thawed sample. Exposing cells to a large volume of hypotonic solution and subsequent washing process was hypothesized to cause lab-induced cell death. Moreover, standard gating strategies must be altered to avoid reporting falsely high viabilities. STUDY DESIGN AND METHODS: We developed a novel method whereby thawed samples were diluted step-wise to 1:2 by 3 additions of 1/3 sample volume using 1% Human Albumin in Dextran 40 (10% Low Molecular Weight Dextran in 0.9% NaCl) separated by 5 min between each addition. An additional 1:10 dilution was required to obtain a desired cell concentration for flow cytometry testing resulting in a 1:20 dilution. RESULTS: Twenty samples were tested simultaneously in a method comparison; the new method demonstrated significant increases in mean cell viabilities for white blood cells, hematopoietic progenitor cells, and T cells as well as reduced standard deviations for each parameter. DISCUSSION: Slow, step-wise dilutions of freshly thawed samples of cryopreserved apheresis products to 1:20 yielded higher and more precise viability measurements compared to quickly washing samples to remove DMSO.

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 aged 60 years 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.

Comparable bacterial growth in platelet concentrates suspended in plasma and platelet additive solution and improved detection of bacterial contamination using a new generation automated culture system.

BACKGROUND: Microbial screening of platelet concentrates (PC) with automated culture methods is widely implemented to reduce septic transfusion reactions. Herein, detection of bacterial contamination in PC was compared between units prepared in plasma and a mix of plasma and platelet additive solution (PAS) and between the BACT/ALERT 3D and next generation BACT/ALERT VIRTUO systems. STUDY DESIGN/METHODS: Double apheresis units were split into single units, diluted in either PAS (PAS-PC) or plasma (plasma-PC), and tested for in vitro quality and sterility prior to spiking with ~30 CFU/unit of Staphylococcus epidermidis, Staphylococcus aureus, Serratia marcescens, and Klebsiella pneumoniae or ~10 CFU/mL of Cutibacterium acnes. Spiked PC were sampled for BACT/ALERT testing (36 and 48 h post-spiking) and colony counts (24, 36, and 48 h post-spiking). Times to detection (TtoD) and bacterial loads were compared between PC products and BACT/ALERT systems (N = 3). RESULTS: Bacterial growth was similar in plasma-PC and PAS-PC. No significant differences in TtoD were observed between plasma-PC and PAS-PC at the 36-h sampling time except for S. epidermidis which grew faster in plasma-PC and C. acnes which was detected earlier in PAS-PC (p < .05). Detection of facultative bacteria was 1.3-2.2 h sooner in VIRTUO compared with 3D (p < .05) while TtoD for C. acnes was not significantly different between the two systems. DISCUSSION: Comparable bacterial detection was observed in plasma-PC and PAS-PC with PC sampling performed at 36-h post blood collection. PC sampling at ≤36 h could result in faster detection of facultative pathogenic organisms with the VIRTUO system and improved PC safety.

Evaluating the effects of hypoxic storage on platelet function and health using a novel storage system.

BACKGROUND: Thousands of units of whole blood (WB) and blood components are transfused daily to treat trauma patients. Improved methods for blood storage are critical to support trauma-related care. The Hemanext ONE® system offers a unique method for hypoxic storage of WB, with successfully demonstrated storage of clinically viable RBCs. This work evaluated the system for the storage of WB, focusing on platelet health and function. STUDY DESIGN AND METHODS: WB was collected from healthy donors and processed through the Hemanext ONE® system. Hemoglobin oxygen saturation (HbSO2) levels of WB were depleted to 10%, 20%, or 30% of total HbSO2 and then stored in PVC bags sealed in oxygen-impermeable bags (except for normoxic control) with samples collected on days 1, 7, and 14 post-processing. Flow cytometry assessed the activation and apoptosis of platelets. Clot dynamics were assessed based on aggregometry and thromboelastography assays, as well as thrombin generation using a calibrated-automated thrombogram method. RESULTS: Hypoxic storage conditions were maintained throughout the storage period. Hypoxia triggered increased lactate production, but pH changes were negligible compared to normoxic control. Storage at 10% HbSO2 had a significant impact on platelet function, resulting in increased activation and reduced clot formation and aggregation. These effects were less significant at 20% and 30% HbSO2. DISCUSSION: This study indicates that platelets are sensitive to hypoxic storage and suffer significant metabolic and functional deterioration when stored at or below 10% HbSO2.

Red Blood Cell Storage with Xenon: Safe or Disruption?

Xenon, an inert gas commonly used in medicine, has been considered as a potential option for prolonged preservation of donor packed red blood cells (pRBCs) under hypoxic conditions. This study aimed to investigate how xenon affects erythrocyte parameters under prolonged storage. In vitro model experiments were performed using two methods to create hypoxic conditions. In the first method, xenon was introduced into bags of pRBCs which were then stored for 42 days, while in the second method, xenon was added to samples in glass tubes. The results of our experiment showed that the presence of xenon resulted in notable alterations in erythrocyte morphology, similar to those observed under standard storage conditions. For pRBC bags, hemolysis during storage with xenon exceeded the acceptable limit by a factor of six, whereas the closed-glass-tube experiment showed minimal hemolysis in samples exposed to xenon. Notably, the production of deoxyhemoglobin was specific to xenon exposure in both cell suspension and hemolysate. However, this study did not provide evidence for the purported protective properties of xenon.

Understanding interactions of plasticisers with a phospholipid monolayer.

The use of DEHP (diethylhexyl phthalate) is now banned for most applications in Europe; the exception is for blood bags, where its toxicity is overshadowed by its ability to extend the storage life of red blood cells. Another plasticiser, BTHC (butanoyl trihexyl citrate), is used in paediatric blood bags but does not stabilise blood cells as effectively. Interactions between plasticisers and lipids are investigated with a phospholipid, DMPC, to understand the increased stability of blood cells in the presence of DEHP as well as bioaccumulation and identify differences with BTHC. Mixed monolayers of DMPC and DEHP or BTHC were studied on Langmuir troughs where surface pressure/area isotherms can be measured. Neutron reflection measurements were made to determine the composition and structure of these mixed layers. A large amount of plasticiser can be incorporated into a DMPC monolayer but once an upper limit is reached, plasticiser is selectively removed from the interface at high surface pressures. The upper limit is found to occur between 40-60 mol% for DEHP and 20-40 mol% for BTHC. The areas per molecule are also different with DEHP being in the range of 50-100 Å2 and BTHC being 65-120 Å2. Results indicate that BTHC does not fit as well as DEHP in DMPC monolayers which could help explain the differences observed with regards to the stability of blood cells.

Platelet dysfunction reversal with cold-stored vs room temperature-stored platelet transfusions.

ABSTRACT: Platelets are stored at room temperature for 5 to 7 days (room temperature-stored platelets [RSPs]). Because of frequent and severe shortages, the US Food and Drug Administration recently approved up to 14-day cold-stored platelets (CSPs) in plasma. However, the posttransfusion function of CSPs is unknown and it is unclear which donors are best suited to provide either RSPs or CSPs. In this study, we sought to evaluate the posttransfusion platelet function and its predictors for platelets stored for the maximum approved storage times (7-day RSPs and 14-day CSPs) in healthy volunteers on acetylsalicylic acid (ASA). We conducted a randomized crossover study in 10 healthy humans. Individuals donated 1 platelet unit, stored at either 22°C or 4°C based on randomization. Before transfusion, participants ingested ASA to inhibit endogenous platelets. Transfusion recipients were tested for platelet function and lipid mediators. Platelet units were tested for lipid mediators only. A second round of transfusion with the alternative product was followed by an identical testing sequence. RSPs reversed platelet inhibition significantly better in αIIbß3 integrin activation-dependent assays. In contrast, CSPs in recipients led to significantly more thrombin generation, which was independent of platelet microparticles. Lysophosphatidylcholine-O species levels predicted the procoagulant capacity of CSPs. In contrast, polyunsaturated fatty acid concentrations predicted the aggregation response of RSPs. In summary, we provide, to our knowledge, the first efficacy data of extended-stored CSPs in plasma. Our results suggest that identifying ideal RSP and CSP donors is possible, and pave the way for larger studies in the future. This trial is registered at www.ClinicalTrials.gov as #NCT0511102.

Effect of leukoreduction on the omics phenotypes of canine packed red blood cells during refrigerated storage.

BACKGROUND: Red blood cell (RBC) storage promotes biochemical and morphological alterations, collectively referred to as storage lesions (SLs). Studies in humans have identified leukoreduction (LR) as a critical processing step that mitigates SLs. To date no study has evaluated the impact of LR on metabolic SLs in canine blood units using omics technologies. OBJECTIVE: Compare the lipid and metabolic profiles of canine packed RBC (pRBC) units as a function of LR in fresh and stored refrigerated (up to 42 days) units. ANIMALS: Packed RBC units were obtained from 8 donor dogs enrolled at 2 different Italian veterinary blood banks. STUDY DESIGN AND METHODS: Observational study. A volume of 450 mL of whole blood was collected using Citrate-Phosphate-Dextrose-Saline-Adenine-Glucose-Mannitol (CPD-SAGM) transfusion bags with a LR filter to produce 2 pRBC units for each donor, without (nLR-pRBC) and with (LR-pRBC) LR. Units were stored in the blood bank at 4 ± 2°C. Sterile weekly samples were obtained from each unit for omics analyses. RESULTS: A significant effect of LR on fresh and stored RBC metabolic phenotypes was observed. The nLR-pRBC were characterized by higher concentrations of free short and medium-chain fatty acids, carboxylic acids (pyruvate, lactate), and amino acids (arginine, cystine). The LR-pRBC had higher concentrations of glycolytic metabolites, high energy phosphate compounds (adenosine triphosphate [ATP]), and antioxidant metabolites (pentose phosphate, total glutathione). CONCLUSION AND CLINICAL IMPORTANCE: Leukoreduction decreases the metabolic SLs of canine pRBC by preserving energy metabolism and preventing oxidative lesions.

Evaluation of platelets componentized with the Reveos Automated Blood Processing System and stored for 7 days at room temperature in a non-Di(2-ethylhexyl) phthalate (DEHP) platelet pooling set.

BACKGROUND: Platelet concentrates (PCs) used for transfusion can be produced by apheresis or derived from whole blood (WB). The Reveos device is the first US Food and Drug Administration-approved automated blood processing system that can produce PCs. In this work, we evaluated the quality and function of Reveos-collected PCs stored for 7 days at room temperature. STUDY DESIGN AND METHODS: WB was collected from healthy donors and componentized on the day of collection (Fresh) or after an overnight hold (Overnight). PCs were produced (n = 7 Fresh; n = 6 Overnight), stored at room temperature in plasma, and evaluated on days 1 and 7 for quality metrics, platelet activation, clot formation, and aggregation response. RESULTS: Platelet count was comparable between Fresh and Overnight PCs. A drop in pH was reported in Fresh day 7 PCs (p < .001, vs. day 1) but not in Overnight. Overnight units displayed the lowest levels of P-selectin expression (p = .0008, vs. day 7 Fresh). Reduced clot strength and increased lysis were observed in both Fresh and Overnight units on day 7 (vs. day 1). Overnight-hold PCs resulted in the highest clot strength on day 7 (p = .0084, vs. Fresh). No differences in aggregation were reported between groups. CONCLUSION: Reveos-processed PCs produced from overnight-hold WB performed better in hemostatic function assays and displayed reduced activation compared to fresh WB-derived PCs, although both PC groups maintained platelet quality throughout storage. Utilization of overnight WB for PC preparation with Reveos holds promise as an alternative method of producing platelets for transfusion purposes.

Platelet activation and blood extracellular vesicles: The influence of venepuncture and short blood storage.

Extracellular vesicles (EVs) as membrane-bound particles released by various cells are potential tools for diagnosis and treatment. Blood cells, particularly platelets, are the source of circulating EVs. MATERIAL: EVs were enriched with gradient ultracentrifugation and measured by nanoparticle tracking assay. A flow cytometric multiplex assay was used for cellular source determination. Activation of platelets was measured as a percentage of CD62p+/CD61+ platelets and correlated with the concentration and size of released EVs. RESULTS: In general there was no statistically significant correlation between EVs` concentration and degree of platelet activation. EVs from different cellular sources were detected. Comparing different needle thicknesses, there was a decrease in the EVs concentration for the 16G needle versus the 21G needle, but no difference was observed for EVs` size and phenotype or platelets activation. During blood storage, platelet activation increased, but there was no effect on the EVs` concentration, size, or phenotype. CONCLUSIONS: Preanalytical factors like needle thickness and storage time can affect the MVs' properties. Activation of platelets during blood collection or blood storage occurs; however, it is difficult to determine its effect on the physiological properties of EVs since the mechanisms of EVs` biogenesis and especially clearness are not precisely known.

Effect of leukoreduction on the metabolism of equine packed red blood cells during refrigerated storage.

BACKGROUND: Understanding of the biochemical and morphological lesions associated with storage of equine blood is limited. OBJECTIVE: To demonstrate the temporal sequences of lipid and metabolic profiles of equine fresh and stored (up to 42 days) and leukoreduced packed red blood cells (LR-pRBC) and non-leukoreduced packed RBC (nLR-pRBC). ANIMALS: Packed RBC units were obtained from 6 healthy blood donor horses enrolled in 2 blood banks. METHODS: Observational study. Whole blood was collected from each donor using transfusion bags with a LR filter. Leukoreduction pRBC and nLR-pRBC units were obtained and stored at 4°C for up 42 days. Sterile weekly sampling was performed from each unit for analyses. RESULTS: Red blood cells and supernatants progressively accumulated lactate products while high-energy phosphate compounds (adenosine triphosphate and 2,3-Diphosphoglycerate) declined. Hypoxanthine, xanthine, and free fatty acids accumulated in stored RBC and supernatants. These lesions were exacerbated in non-LR-pRBC. CONCLUSION AND CLINICAL IMPORTANCE: Leukoreduction has a beneficial effect on RBC energy and redox metabolism of equine pRBC and the onset and severity of the metabolic storage lesions RBC.

Estimated median density identifies donor age and sex differences in red blood cell biological age.

BACKGROUND: Donors possess heterogeneous red cell concentrates (RCCs) in terms of the biological age of their red blood cells (RBCs) as a direct result of various donor-dependent factors influencing rates of erythropoiesis. This study aimed to estimate the median biological age of RBCs in RCCs based on donor age and sex to investigate inherent differences in blood products' biological ages over hypothermic storage using estimated median densities (EMDs). STUDY DESIGN: Sixty RCCs were collected from four donor groups; male and female teenagers (17-19 years old) and seniors (75+ years old). A Percoll density-based separation approach was used to quantify the EMDs indicative of biological age. EMD and mean corpuscular hemoglobin (MCHC) were compared by correlation analyses. RESULTS: Differences in the median biological age of RCC units were observed with male donors having significantly higher EMDs compared to females (p < .001). Teen male donors possessed the highest EMDs with significantly elevated levels of biologically aged RBCs compared to both female donor groups, regardless of storage duration (p < .05). Throughout most of the 42-day storage period, senior donors, particularly senior females, demonstrated the strongest correlation between EMD and MCHC (R2 > 0.5). CONCLUSIONS: This study provides further evidence that there are inherent differences between the biological age profiles of RBCs between blood donors of different sex and age. Our findings further highlight that biological age may contribute to RBC quality during storage and that donor characteristics need to be considered when evaluating transfusion safety and efficacy.

Impact of production methods and storage conditions on extracellular vesicles in packed red blood cells and platelet concentrates.

The use of blood and blood products can be life-saving, but there are also certain risks associated with their administration and use. Packed red blood cells (pRBCs) and platelet concentrates are the most commonly used blood products in transfusion medicine to treat anemia or acute and chronic bleeding disorders, respectively. During the production and storage of blood products, red blood cells and platelets release extracellular vesicles (EVs) as a result of the storage lesion, which may affect product quality. EVs are subcellular structures enclosed by a lipid bilayer and originate from the endosomal system or from the plasma membrane. They play a pivotal role in intercellular communication and are emerging as important regulators of inflammation and coagulation. Their cargo and their functional characteristics depend on the cell type from which they originate, as well as on their microenvironment, influencing their capacity to promote coagulation and inflammatory responses. Hence, the potential involvement of EVs in transfusion-related adverse events is increasingly recognized and studied. Here, we review the knowledge regarding the effect of production and storage conditions of pRBCs and platelet concentrates on the release of EVs. In this context, the mode of processing and anticoagulation, the influence of additive solutions and leukoreduction, as well as the storage duration will be addressed, and we discuss potential implications of EVs for the clinical outcome of transfusion.

Preservation of recipient plasma sphingosine-1-phosphate levels reduces transfusion-related acute lung injury.

Cold-stored (CS) platelets are once again being reintroduced for clinical use. Transfused CS platelets offer benefits over room temperature-stored (RTS) platelets such as increased hemostatic effects and prolongation of shelf-life. Despite these advantages little is known about their association with transfusion-related acute lung injury (TRALI). TRALI is associated with prolonged storage of RTS platelets and has a mortality of >15%. Determining the safety of CS platelets is important considering their proposed use in TRALI-vulnerable populations with inflammation such as surgical patients or patients with trauma. Donor platelet-derived ceramide causes TRALI, whereas donor platelet sphingosine-1-phosphate (S1P) is barrier protective. Females have higher plasma levels of S1P than males. Cold temperatures increase S1P levels in cells. Therefore, we hypothesized that female (donors or recipients) and/or CS platelets would decrease TRALI. To test this, we compared how male and female donor and recipient allogeneic platelet transfusions of CS (4°C) versus RTS (23°C) platelets stored for 5 days influence murine TRALI. Transfusion of CS platelets significantly reduced recipient lung tissue wet-to-dry ratios, bronchoalveolar lavage total protein, lung tissue myeloperoxidase enzyme activity, histological lung injury scores, and increased plasma sphingosine-1-phosphate (S1P) levels compared with RTS platelet transfusions. Female as opposed to male recipients had less TRALI and higher plasma S1P levels. Female donor mouse platelets had higher S1P levels than males. Mouse and human CS platelets had increased S1P levels compared with RTS platelets. Higher recipient plasma S1P levels appear protective considering females, and males receiving platelets from females or male CS platelets had less TRALI.NEW & NOTEWORTHY Transfusion-related acute lung injury (TRALI) though relatively rare represents a severe lung injury. The sphingolipid sphingosine-1-phosphate (S1P) regulates the severity of platelet-mediated TRALI. Female platelet transfusion recipient plasmas or stored platelets from female donors have higher S1P levels than males, which reduces TRALI. Cold storage of murine platelets preserves platelet-S1P, which reduces TRALI in platelet-transfused recipients.

Performance of far forward iceless blood storage containers in controlled cold environments.

BACKGROUND: The Golden Hour Box (GHB), an iceless blood container designed for transfusion closest to the point of injury, is used by military medical teams in remote damage control resuscitation. While its performance is well-established in hot environments, it remains underexplored in cold conditions, a significant consideration in emerging global conflict zones. STUDY DESIGN AND METHODS: Four GHBs were preconditioned at +4°C or +18°C for 8 h and subsequently exposed to controlled laboratory simulated temperatures of -5, -15, and -25°C for 100 h. The study focused on their capability to maintain an internal temperature between +2 and +6°C, the recommended range for red blood cells unit storage and transport, using calibrated sensors for precise monitoring. RESULTS: When exposed to negative Celsius temperatures, GHBs showed varied performance depending on preconditioning temperatures. When preconditioned at +4°C, GHBs maintained an internal temperature within the target range (+2 to +6°C) for 100 h at -5°C, 52 ± 1 h at -15°C, and 29 ± 4 h at -25°C. In contrast, the internal temperature of GHBs preconditioned at +18°C exceeded this range in less than 30 min, then dropped below 2°C more rapidly than those preconditioned at +4°C, occurring within 20 ± 2 h at -15 and 13 ± 1 h at -25°C. CONCLUSION: The GHB, when properly preconditioned, effectively maintains internal temperatures suitable for blood product transport in extreme cold. Future research, including analyses of blood performances, is still needed to validate these results in more realistic operational conditions for use in cold environments.

Effects of blood sample storage time, temperature, anti-coagulants and blood stabiliser on lymphocyte phenotyping.

Medical diagnostic laboratories have come under further scrutiny to ensure quality standards of their service and external quality assurance (EQA) programs involving multiple laboratories have been used to gauge this quality based on a consensus. However, because of the geographical distances within a country or internationally, cell surface marker expressions may change due to time delays and transport temperatures. Attention was given to this issue some decades ago and hence requires a re-evaluation in consideration of updated methods, reagents and instruments for flow cytometry and phenotyping. We have undertaken an extensive study to examine the effects of various conditions on blood storage akin to that experienced by patient samples as well as EQA programs, examining expression of lymphocyte surface markers, CD3, CD4, CD8, CD2, CD19, CD20, CD16/56 and HLA-DR. Assessment of lithium-heparin anticoagulated whole blood showed an increase in percentage of CD3+ and CD8+ T cells and a decrease in CD16/56+ NK cells after storage at room temperature (RT) for 24 and/or 48 h. In comparison, storage at 4°C led to a decrease in percentage of CD4+ and increase in percentage of CD8+ cells. The low temperature also caused an increase in percentage of B cells (CD19+, CD20+). While storage at RT did not alter levels of HLA-DR+ CD3+ T cells, there was a significant increase in percentage of these cells after 48 h. Changes were also seen at both temperatures when EDTA was used as an anti-coagulant. Assessment of blood treated with a stabiliser, normally used in the EQA samples (Streck Cell Preservative), reduced the range of lymphocyte subsets affected, with only CD2+ and CD20+ cells being significantly different at both temperatures, We conclude that 24-48 h storage/transport can affect the percentage of CD3+, CD4+ T cells, CD8+ T cells, B cells, NK cells and HLADR+ T cells which can be minimised by using the blood stabiliser as per EQA programs and we emphasise the need to adopt this in the processing of patients' blood samples.

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

BACKGROUND AND OBJECTIVES: 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. MATERIALS AND METHODS: 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. RESULTS: 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. CONCLUSION: 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.