Modification of deglycerolization procedure improves processing and post-thaw quality of cryopreserved sickle trait red cell concentrates.

Red blood cell (RBC) transfusion is a critical therapy for those with sickle cell disease (SCD). Alloimmunization is frequent for those with SCD and may limit the availability of matched RBC. Cryopreserved RBCs, from family members or donors with a similar RBC antigen profile could provide a viable alternative to avoid further alloimmunization and prevent hemolytic transfusion-related events. However, cryopreserved SCD and Sickle Cell trait (S-trait) donor RBC units suffer from reduced recovery following deglycerolization. This study proposes and tests a modified deglycerolization protocol using an automated cell processor to mitigate RBC loss. Six red cell concentrates (RCC) from donors with S-trait and six control RCCs were glycerolized, frozen (<-65 °C) and deglycerolized on the ACP 215 using modified parameters (decreased hypertonic solution flow rate (100 mL/min) and hypertonic equilibration delay (120 s), and increased NaCl dilution volumes (500 mL). Quality testing included: hematocrit (HCT), hemolysis, indices, extracellular potassium, morphology, osmotic fragility, osmotic gradient ektacytometry, hemoglobin (HGB), and recovery. Canadian standards (CS) indicate that acceptable deglycerolized units for transfusion require a HCT ≤0.80 L/L, HGB ≥35 g/unit, and hemolysis <0.8 % in 90 % of units tested. No significant differences in HGB or RBC recovery were observed between study groups. Significant differences between study groups were identified in osmotic fragility and osmotic gradient ektacytometry parameters. Of the 6 S-trait RCCs, 3/6 units were within the HCT, HGB and hemolysis thresholds set by the CS. The modified deglycerolization protocol provides a path for the routine cryopreservation of S-trait RBCs.

Advancing in vivo assessment of red blood cell transfusions: A call for radiation-free methods in transfusion medicine.

RBC transfusions are a vital clinical therapy to treat anemic patients. The in vivo assessment of red blood cell (RBC) quality post-transfusion is critical to ensuring that the introduction of new RBC products meet established regulatory and clinical quality requirements. Although in vitro quality control testing is routinely performed by blood manufacturers, it is crucial that in vivo tests are performed during the evaluation and regulatory process of new RBC products. This article reviews existing in vivo techniques, like chromium-51 labelling and biotinylation, for determining the circulation and survival of RBCs, and advocates for a move to radiation-free methods. The timely need for radiation-free methods to assess emerging non-DEHP container systems is just one example of why efforts to improve the methods available for in vivo quality assessment is important in transfusion medicine. This review aims to advance our understanding of RBC transfusion in vivo quality assessment and enhance transfusion practices.

Comparing two extracellular additives to facilitate extended storage of red blood cells in a supercooled state.

Background: Adenosine triphosphate (ATP) levels guide many aspects of the red blood cell (RBC) hypothermic storage lesions. As a result, efforts to improve the quality of hypothermic-stored red cell concentrates (RCCs) have largely centered around designing storage solutions to promote ATP retention. Considering reduced temperatures alone would diminish metabolism, and thereby enhance ATP retention, we evaluated: (a) whether the quality of stored blood is improved at -4°C relative to conventional 4°C storage, and (b) whether the addition of trehalose and PEG400 can enhance these improvements. Study Design and Methods: Ten CPD/SAGM leukoreduced RCCs were pooled, split, and resuspended in a next-generation storage solution (i.e., PAG3M) supplemented with 0-165 mM of trehalose or 0-165 mM of PEG400. In a separate subset of samples, mannitol was removed at equimolar concentrations to achieve a fixed osmolarity between the additive and non-additive groups. All samples were stored at both 4°C and -4°C under a layer of paraffin oil to prevent ice formation. Results: PEG400 reduced hemolysis and increased deformability in -4°C-stored samples when used at a concentration of 110 mM. Reduced temperatures did indeed enhance ATP retention; however, in the absence of an additive, the characteristic storage-dependent decline in deformability and increase in hemolysis was exacerbated. The addition of trehalose enhanced this decline in deformability and hemolysis at -4°C; although, this was marginally alleviated by the osmolarity-adjustments. In contrast, outcomes with PEG400 were worsened by these osmolarity adjustments, but at no concentration, in the absence of these adjustments, was damage greater than the control. Discussion: Supercooled temperatures can allow for improved ATP retention; however, this does not translate into improved storage success. Additional work is necessary to further elucidate the mechanism of injury that progresses at these temperatures such that storage solutions can be designed which allow RBCs to benefit from this diminished rate of metabolic deterioration. The present study suggests that PEG400 could be an ideal component in these solutions.

Extracellular vesicles: effectors of transfusion-related acute lung injury.

Respiratory transfusion reactions represent some of the most severe adverse reactions related to receiving blood products. Of those, transfusion-related acute lung injury (TRALI) is associated with elevated morbidity and mortality. TRALI is characterized by severe lung injury associated with inflammation, pulmonary neutrophil infiltration, lung barrier leak, and increased interstitial and airspace edema that cause respiratory failure. Presently, there are few means of detecting TRALI beyond clinical definitions based on physical examination and vital signs or preventing/treating TRALI beyond supportive care with oxygen and positive pressure ventilation. Mechanistically, TRALI is thought to be mediated by the culmination of two successive proinflammatory hits, which typically comprise a recipient factor (1st hit-e.g., systemic inflammatory conditions) and a donor factor (2nd hit-e.g., blood products containing pathogenic antibodies or bioactive lipids). An emerging concept in TRALI research is the contribution of extracellular vesicles (EVs) in mediating the first and/or second hit in TRALI. EVs are small, subcellular, membrane-bound vesicles that circulate in donor and recipient blood. Injurious EVs may be released by immune or vascular cells during inflammation, by infectious bacteria, or in blood products during storage, and can target the lung upon systemic dissemination. This review assesses emerging concepts such as how EVs: 1) mediate TRALI, 2) represent targets for therapeutic intervention to prevent or treat TRALI, and 3) serve as biochemical biomarkers facilitating TRALI diagnosis and detection in at-risk patients.

Immunological impact of the CD71+ RBCs: A potential immune mediator in transfusion.

Donor - recipient sex - mismatched transfusion is associated with increased mortality. The mechanisms for this are not clear, but it may relate to transfusion-related immunomodulation. Recently, CD71+ erythroid cells (CECs), including reticulocytes (CD71+ RBCs) and erythroblasts, have been identified as potent immunoregulatory cells. The proportion of CD71+ RBCs in the peripheral blood is sufficient to play a potential immunomodulatory role. Differences in the quantity of CD71+ RBCs are dependent on blood donor sex. The total number of CD71+ RBCs in red cell concentrates is also affected by blood manufacturing methods, and storage duration. As a component of the total CECs, CD71+ RBCs can affect innate and adaptive immune cells. Phagocytosed CECs directly reduce TNF-α production from macrophages. CECs can also suppress the production of TNF-α production from antigen presenting cells. Moreover, CECs can suppress T cell proliferation thorough immune mediation and / or direct cell-to-cell interactions. Different in their biophysical features compared to mature RBCs, blood donor CD71+ RBCs may be preferential targets for the macrophages. This report summarizes the currently literature supporting an important role for CD71+ RBCs in adverse transfusion reactions including immune mediation and sepsis.

An evaluation of the clinical impacts of 7-day platelets.

BACKGROUND: In August 2017, Canadian Blood Services extended the shelf-life of platelet concentrates from 5 to 7 days. The clinical impacts of this policy change remain unclear. STUDY DESIGN AND METHODS: We used a before-after retrospective design of platelet-transfused adult inpatients in Hamilton, ON, Canada. Data were captured for 18 months before (Period 1: February 2016-July 2017) and 18 months after (Period 2: September 2017-February 2019) 7-day platelet implementation. Primary outcome was absolute platelet count increment (ACI) in univariate and multivariate analyses adjusted for confounders. Data were obtained from our institution's transfusion database, Ontario's Transfusion Transmitted Injuries Surveillance System, and the blood supplier. RESULTS: Overall, 1360 patients with single dose platelet transfusions were included in Period 1 and 1211 patients in Period 2. Median age at admission was 66 years, and approximately 40% of patients underwent cardiac surgery. Using a non-inferiority margin of -10 × 109 /L, platelets transfused during the 7-day storage period were non-inferior to those transfused in the 5-day storage period [mean count difference - 4.63 × 109 /L (95% CI -7.40 to -1.87, p = 0.0001)]. However, platelet ACIs following transfusion consistently trended lower in the 7-day group for all patients and subgroups. No differences in secondary clinical outcomes were observed. Platelet expiry reduced from 8.1 to 6.3% (p < 0.0001). CONCLUSION: Platelet transfusions following 7-day storage policy were non-inferior to transfusions in the 5-day policy period, although reduced ACIs were observed. There were no increases in adverse clinical outcomes.

Closed system processing variables affect post-thaw quality characteristics of cryopreserved red cell concentrates.

BACKGROUND: Differences in manufacturing conditions using the Haemonetics ACP 215 cell processor result in cryopreserved red cell concentrates (RCCs) of varying quality. This work studied the effect of processing method, additive solution, and storage duration on RCC quality to identify an optimal protocol for the manufacture of cryopreserved RCCs. MATERIALS AND METHODS: RCCs were pooled-and-split and stored for 7, 14, or 21 days before cryopreservation. Units were glycerolized with the ACP 215 using a single or double centrifugation method. After thawing, the RCCs were deglycerolized, suspended in AS-3, SAGM, ESOL, or SOLX/AS-7, and stored for 0, 3, 7, 14, or 21 days before quality testing. Quality assessments included hemoglobin content, hematocrit, hemolysis, adenosine triphosphate (ATP), supernatant potassium, and mean cell volume. RESULTS: Both glycerolization methods produced RCCs that met regulatory standards for blood quality. Dual centrifugation resulted in higher hemoglobin content, fewer processing alerts, and a shorter deglycerolization time than single centrifugation processing. Units processed with AS-3 and ESOL met regulatory standards when stored for up to 21 days pre-cryopreservation and 21 days post-deglycerolization. However, ESOL demonstrated superior maintenance of ATP over RBCs in AS-3. Some RCCs suspended in SAGM and SOLX exceeded acceptable hemolysis values after 7 days of post-deglycerolization storage regardless of pre-processing storage length. CONCLUSIONS: When manufacturing cryopreserved RCCs using the ACP 215, dual centrifugation processing with AS-3 or ESOL additive solutions is preferred, with storage periods of up to 21 days both pre-processing and post-deglycerolization.

Automated closed volume reduction process for apheresis stem cell grafts: From development to clinical implementation.

BACKGROUND: Collection of HPC by apheresis (HPC-A) can sometimes result in higher collection volumes, increasing the dimethyl sulfoxide (DMSO) volume infused into patients and the space requirements in liquid nitrogen freezers. Volume reduction prior to the addition of cryoprotectant is an efficient means to reduce the DMSO load infused into patients and to optimize freezer storage space. STUDY DESIGN AND METHODS: To implement a closed semi-automated volume reduction process, a method was developed to produce leukocyte-rich mock apheresis products using buffy coats derived from whole blood collections. The mock HPC products were then used to measure the efficiency and reliability of the semi-automated process over a range of volumes and cell concentrations. The resulting data was used to support the implementation of the process with concurrent monitoring. RESULTS: A closed, semi-automated volume reduction process resulted in recoveries of over 93% and 91% of white blood cells and CD34+ cells with no significant loss of product viability or potency. Mean doses of CD34+ and CFU infused per kilogram recipient body weight were 4.0 ± 1.1 × 106 /kg and 4.2 ± 1.7 × 105 /kg, resulting in no delays in median time to neutrophil and platelet engraftment, significant increase in adverse reaction or nonconformances. DISCUSSION: The effectiveness outcomes of the first Canadian experience in the implementation of a closed semi-automated volume reduction system in the processing of HPC-A products for autologous transplant have met the predetermined acceptance criteria, supporting its use in a stem cell manufacturing laboratory compliant with good manufacturing practice regulations.

Preparing small-dose red cell concentrates (RCCs) for neonatal and pediatric transfusions: Impact of RCC volume, storage, and irradiation.

BACKGROUND: Preparing small-dose red cell concentrates (RCCs) is a common practice for pediatric and neonatal transfusions. However, there is a lack of quality monitoring data to indicate that both the preparation and storage of small-dose RCCs does not alter in vitro red cell quality. The present study seeks to provide data to support this practice. MATERIALS AND METHODS: To evaluate quality of stored small aliquots, six ABO/Rh matched leukoreduced citrate phosphate-dextrose/saline-adenine-glucose-mannitol (LR CPD/SAGM) RCCs were pooled and split into 30 ml aliquots, 80 ml aliquots, and a standard 290 ml unit, with testing performed for up to 43 days post-collection. To evaluate the impact of irradiation on small-dose RCC preparation, a total of 48 independent LR CPD/SAGM RCCs were used (non-irradiated: n = 24; irradiated: n = 24). Aliquoting with/without irradiation was performed within 7 days of collection and baseline testing was performed within 24 h of aliquot production. RESULTS: Limited variability in hemolysis, mean cell volume, and extracellular potassium concentrations were seen between the different aliquot sizes throughout the 43-day storage period. Aliquot production did not accentuate damage based on any of these tested parameters in both the non-irradiated and irradiated subsets. A significant increase was seen in the potassium concentrations in the irradiated parent and aliquot samples relative to their non-irradiated counterparts. CONCLUSIONS: Non-irradiated small-aliquot dose RCCs meet in vitro quality criteria required for safe transfusion throughout the 42-day storage period. The same can be said for aliquots derived from irradiated units and tested within 24 h of aliquot production.

The Effect of Sex-Mismatched Red Blood Cell Transfusion on Endothelial Cell Activation in Critically Ill Patients.

Background: Observational studies suggest that sex-mismatched transfusion is associated with increased mortality. Mechanisms driving mortality are not known but may include endothelial activation. The aim of this study is to investigate the effects of sex-mismatched red blood cell (RBC) transfusions on endothelial cell activation markers in critically ill patients. Study Design and Methods: In patients admitted to the intensive care unit who received a single RBC unit, blood samples were drawn before (T0), 1 h after (T1), and 24 h after transfusion (T24) for analysis of soluble syndecan-1, soluble intercellular adhesion molecule-1, soluble thrombomodulin (sTM), von Willebrand factor antigen, interleukin-6 (IL-6), and tumor necrosis factor-alpha (TNFα). Changes in the levels of these factors were compared between sex-matched and sex-mismatched groups. Results: Of 69 included patients, 32 patients were in the sex-matched and 37 patients were in the sex-mismatched group. Compared to baseline, sex-matched transfusion was associated with significant reduction in sTM level (p value = 0.03). Between-group comparison showed that levels of syndecan-1 and sTM were significantly higher in the sex-mismatched group compared to the sex-matched group at T24 (p value = 0.04 and 0.01, respectively). Also, TNFα and IL-6 levels showed a statistically marginal significant increase compared to baseline in the sex-mismatched group at T24 (p value = 0.06 and 0.05, respectively), but not in the sex-matched group. Discussion: Transfusion of a single sex-mismatched RBC unit was associated with higher syndecan-1 and sTM levels compared to transfusion of sex-matched RBC unit. These findings may suggest that sex-mismatched RBC transfusion is associated with endothelial activation.

The response of a human haematopoietic cell line to trehalose-loaded liposomes and their effect on post-thaw membrane integrity.

Dimethyl sulphoxide (DMSO) used in haematopoietic stem cell (HSC) cryopreservation has been linked to an increased incidence of adverse reactions following transplantation. In the interest of reducing the required DMSO concentrations, we have evaluated the use of unilamellar liposomes to internalize the non-toxic, cell-impermeable disaccharide, trehalose into HSCs and characterized the cryoprotective efficacy of this strategy. A fluorescent marker, 5(6)-carboxyfluorescein (200 µmol/L), was used for trehalose internalization following a 5 h incubation at 37 °C with liposome concentrations ranging from 0.5 mM to 4 mM. Cells were frozen (1 °C/min to -80 °C) following treatment with either 3 mM or 4 mM of liposomes (5 h, 37 °C) containing 0.2 mol/L trehalose either in the presence or absence of 0.2 mol/L extracellular trehalose. Increasing the liposome concentration from 3 mM to 4 mM corresponded to a significant (p = 0.046) increase in the mean fluorescent intensity (MFI) (3 mM 512 ± 7.07; 4 mM: 916 ± 28.3). Post-thaw membrane integrity indicated that the presence of trehalose both inside and outside when internalized using a liposome concentration of 4 mM significantly improved survival relative to the sole presence of extracellular trehalose (p = 0.02). However, viability was diminished relative to a standard DMSO control (trehalose: 32.5% ± 1.7%; DMSO: 85.0% ± 4.6%). This study confirms that the protective efficacy of trehalose is enhanced when it is present on both sides of the membrane; however, it reinforces concerns surrounding the efficiency of using liposomes as a vehicle to transfer trehalose into cells.

in Vitro Effect of Methamphetamine on Proliferation, Differentiation and Apoptosis of Adipose Tissue Stem Cells.

PURPOSE: Among abused substances, methamphetamine is a psychostimulant drug widely used recreationally with public health importance. This study investigated the effect of methamphetamine on proliferation, differentiation, and apoptosis of human adipose tissue stem cells (AdSCs). METHODS: AdSCs were isolated from human abdominal adipose tissue and were characterized for mesenchymal properties and growth kinetics. MTT assay was undertaken to assess methamphetamine toxicity on proliferation and differentiation properties and apoptosis of hAdSCs. RESULTS: Isolated cells were shown to have mesenchymal properties and a population doubling time (PDT) of 40.1 h. Following methamphetamine treatment, expressions of KI-67 and TPX2 as proliferation genes and Col1A1 and PPARg as differentiation genes decreased. Methamphetamine administration increased the expression of Bax and decreased Bcl-2 genes responsible for apoptosis. CONCLUSIONS: Our data suggested when AdSCs were exposed to methamphetamine, it decreased proliferation and differentiation properties of stem cells together with an increase in apoptosis. These findings can be added to the literature, especially when methamphetamine is used recreationally for weight loss purposes.

Copro-polymerase chain reaction has higher sensitivity compared to centrifugal fecal flotation in the diagnosis of taeniid cestodes, especially Echinococcus spp, in canids.

Prompt and reliable diagnostic tests for taeniid infection in canids are important due to the risk of zoonoses like Echinococcus spp. Current diagnostic methods relying on fecal flotation lack sensitivity and specificity, but this has rarely been quantified due to the challenges in performing adult cestode recovery (the gold standard) in domestic dogs (Canis familiaris). Therefore, we recovered adult Taenia and Echinococcus spp. from intestines, as well as fecal/intestinal material from 484 wild canids trapped for fur in two Canadian provinces (276 foxes - primarily Vulpes vulpes, coyotes - Canis latrans, and wolves - Canis lupus in Québec and 208 coyotes in Saskatchewan). The performances of a newly developed coproPCR for tapeworm DNA detection in dogs, and centrifugal fecal flotation using Sheather's solution, were evaluated against adult cestode recovery. Overall, adult taeniid cestode prevalence (Taenia and/or Echinococcus) was 28 % (95 % CI: 23-33 %) in Québec (62 % (CI: 51-73%) of 74 coyotes, 65 % (CI: 44-82) of 23 wolves, and 11 % (CI: 7-16%) of 179 foxes) and 79 % (CI: 73-84%) of 208 coyotes in Saskatchewan. In Québec, E. canadensis and Taenia spp. were detected in coyotes and wolves, and foxes were only infected with Taenia spp., whereas Saskatchewan coyotes were predominantly infected with E. multilocularis (at significantly higher prevalence, but not intensity, than coyotes in Québec). Compared with centrifugal fecal flotation, the new coproPCR had at least double the sensitivity (58 % vs 23 % in QC coyotes, 57 % vs 23 % in QC wolves, 24 % vs 0% in QC foxes, and 80 % vs 25 % in SK coyotes). Notably, no taeniid eggs were detected on flotations from foxes infected with Taenia spp., and the new coproPCR had highest sensitivity in Saskatchewan coyotes, which were predominantly infected with E. multilocularis. CoproPCR has promising prospects for use in Veterinary clinics and diagnostic laboratories to detect taeniid cestode infections because of its higher sensitivity than faecal flotation methods. This is particularly important for zoonotic Echinococcus spp. where, from a public health perspective, false negatives are a much greater concern than false positives.

Proliferative and Regenerative Effect of Acetonic Extract of Feijoa sellowiana on Stem Cells.

BACKGROUND: Feijoa is widely used in medicine due to their anti-inflammatory, antioxidant, antimicrobial and antitumor properties. The current investigation studied the proliferative and regenerative effect of acetonic extract of Feijoa sellowiana on stem cells. METHODS: Acetone extract of Feijoa was prepared using percolator and rotary machines. Human bone marrow stem cells (hBMSCs) were used as experimental in vitro model and characterized morphologically, by flowcytometry, and differentiation properties. The toxicity of the extract on hBMSCs was determined by MTT assay. The viability and growth kinetics of hBMSCs treated to Feijoa was determined. Real time PCR was used for changes in expression of proliferative and apoptotic genes on day 7th. RESULTS: MTT assay demondtrated that Feijoa at doses less than 200 ng/ml did not show any cytotoxic effect on hBMSCs and increased the cell proliferation until day 3rd followed by a non-significant slow decreasing trend until day 7th. Population doubling time (PDT) showed a decline until day 3rd followed by an increase until day 7th. A significant rise in expression of Bax and decline in Bcl-2 expression were noted on day 7th. CONCLUSION: The modulatory activity of Feijoa may be responsible for its increasing effect on cell proliferation till day 3rd. Therefore, when faster proliferation during a shorter time period is targeted, Feijoa can be safely added to the culture media in the first three days.

Cryoprotectant-dependent control of intracellular ice recrystallization in hepatocytes using small molecule carbohydrate derivatives.

To promote the recovery of cells that undergo intracellular ice formation (IIF), it is imperative that the recrystallization of intracellular ice is minimized. Hepatocytes are more prone to IIF than most mammalian cells, and thus we assessed the ability of novel small molecule carbohydrate-based ice recrystallization inhibitors (IRIs) to permeate and function within hepatocytes. HepG2 monolayers were treated with N-(4-chlorophenyl)-d-gluconamide (IRI 1), N-(2-fluorophenyl)-d-gluconamide (IRI 2), or para-methoxyphenyl-ß-D-glycoside (IRI 3) and fluorescent cryomicroscopy was used for real time visualization of intracellular ice recrystallization. Both IRI 2 and IRI 3 reduced rates of intracellular recrystallization, whereas IRI 1 did not. IRI 2 and IRI 3, however, demonstrated a marked reduction in efficiency in the presence of the most frequently used permeating cryoprotectants (CPAs): glycerol, propylene glycol (PG), dimethyl sulfoxide (DMSO), and ethylene glycol (EG). Nevertheless, IRI 3 reduced rates of intracellular recrystallization relative to CPA-only controls in the presence of glycerol, PG, and DMSO. Interestingly, IRI preparation in trehalose, a commonly used non-permeating CPA, did not impact the activity of IRI 3. However, trehalose did increase the activity of IRI 1 while decreasing that of IRI 2. While this study suggests that each of these compounds could prove relevant in hepatocyte cryopreservation protocols where IIF would be prominent, CPA-mediated modulation of intracellular IRI activity is apparent and warrants further investigation.

Transient loss of membrane integrity following intracellular ice formation in dimethyl sulfoxide-treated hepatocyte and endothelial cell monolayers.

Immediate post-thaw evaluation of membrane integrity has proven to yield overestimates of cell survival under conditions that preclude intracellular ice formation (IIF). However, prominent theories on the mechanisms of intracellular nucleation suggest a damaged membrane can reseal, prompting us to evaluate whether immediate post-thaw assessments of membrane integrity can in fact underestimate cell survival under conditions that promote IIF. HUVEC and HepG2 monolayers were treated with 1.4 M DMSO and frozen to -25 °C under conditions that formed either 0% or 100% IIF. Membrane integrity was evaluated both immediately and 24 h post-thaw, with metabolic activity assessments performed 24 h post-thaw as a secondary measure of survival. Treatment with 1.4 M DMSO and nucleation of 100% IIF resulted in a drastic increase in the relative percent of membrane intact cells following a 24 h culture period (HUVEC: 90.2% ± 0.7%; HepG2: 70.4% ± 4.0%), which correlated with 24 h post-thaw metabolic activity. These differences between the immediate and 24 h post-thaw membrane integrity assessments were significantly more than those seen in the absence of either IIF or DMSO treatment. Therefore, a high incidence of IIF in DMSO-treated monolayers may lead to erroneous underestimates of cell survival when conducting immediate post-thaw assessments of membrane integrity.

Objective assessment of stored blood quality by deep learning.

Stored red blood cells (RBCs) are needed for life-saving blood transfusions, but they undergo continuous degradation. RBC storage lesions are often assessed by microscopic examination or biochemical and biophysical assays, which are complex, time-consuming, and destructive to fragile cells. Here we demonstrate the use of label-free imaging flow cytometry and deep learning to characterize RBC lesions. Using brightfield images, a trained neural network achieved 76.7% agreement with experts in classifying seven clinically relevant RBC morphologies associated with storage lesions, comparable to 82.5% agreement between different experts. Given that human observation and classification may not optimally discern RBC quality, we went further and eliminated subjective human annotation in the training step by training a weakly supervised neural network using only storage duration times. The feature space extracted by this network revealed a chronological progression of morphological changes that better predicted blood quality, as measured by physiological hemolytic assay readouts, than the conventional expert-assessed morphology classification system. With further training and clinical testing across multiple sites, protocols, and instruments, deep learning and label-free imaging flow cytometry might be used to routinely and objectively assess RBC storage lesions. This would automate a complex protocol, minimize laboratory sample handling and preparation, and reduce the impact of procedural errors and discrepancies between facilities and blood donors. The chronology-based machine-learning approach may also improve upon humans' assessment of morphological changes in other biomedically important progressions, such as differentiation and metastasis.

Trehalose-Based Polyethers for Cryopreservation and Three-Dimensional Cell Scaffolds.

The capability to slow ice growth and recrystallization is compulsory in the cryopreservation of cells and tissues to avoid injuries associated with the physical and chemical responses of freezing and thawing. Cryoprotective agents (CPAs) have been used to restrain cryoinjury and improve cell survival, but some of these compounds pose greater risks for the clinical application of cryopreserved cells due to their inherent toxicity. Trehalose is known for its unique physicochemical properties and its interaction with the phospholipids of the plasma membrane, which can reduce cell osmotic stress and stabilized the cryopreserved cells. Nonetheless, there has been a shortage of relevant studies on the synthesis of trehalose-based CPAs. We hereby report the synthesis and evaluation of a trehalose-based polymer and hydrogel and its use as a cryoprotectant and three-dimensional (3D) cell scaffold for cell encapsulation and organoid production. In vitro cytotoxicity studies with the trehalose-based polymers (poly(Tre-ECH)) demonstrated biocompatibility up to 100 mg/mL. High post-thaw cell membrane integrity and post-thaw cell plating efficiencies were achieved after 24 h of incubation with skin fibroblast, HeLa (cervical), and PC3 (prostate) cancer cell lines under both controlled-rate and ultrarapid freezing protocols. Differential scanning calorimetry and a splat cooling assay for the determination of ice recrystallization inhibition activity corroborated the unique properties of these trehalose-based polyethers as cryoprotectants. Furthermore, the ability to form hydrogels as 3D cell scaffolds encourages the use of these novel polymers in the development of cell organoids and cryopreservation platforms.

Platelet vesicles are potent inflammatory mediators in red blood cell products and washing reduces the inflammatory phenotype.

BACKGROUND: Studies suggest that washing red cell concentrates (RCCs) to remove soluble mediators and/or inflammatory components, such as extracellular vesicles (EVs), may lead to better clinical outcomes. This study tested the hypothesis that non-red blood cell (RBC) generated vesicles in RCC are potent inflammatory mediators in vitro and washing RCCs can reduce these vesicles and subsequently decrease the inflammatory activity of RCCs. STUDY DESIGN AND METHODS: Sixteen RCCs were pooled and split into four groups based on pre-wash storage time (Day 2 or 14; n = 4/group). Each group was tested 24 hours and 7 days post-wash. Characteristics of RBCs and EVs, cytokines released by monocytes, and expression of human umbilical vein endothelial cells (HUVECs) adhesion molecules were assessed. RESULTS: All RCCs meet quality standards for hemolysis, hematocrit, and hemoglobin. Washing did not remove residual platelets from RCCs but led to a significant reduction in platelet-EV count regardless of the group. Supernatant of RCCs washed on Day 14 and stored for 24 hours had significantly lower concentrations of RBC-EVs and white blood cell EVs compared to unwashed controls. Supernatant of unwashed RCCs showed higher production of inflammatory cytokines/chemokines MCP-1, IL-8, and TNF-α, and heightened expression of HUVEC VCAM-1, which were significantly reduced by washing. Spiking washed RCC supernatants with platelet-EVs showed significant increase in IL-8, MCP-1, VCAM-1, and E-selection in groups washed on Day 14. CONCLUSIONS: Platelet-EVs in RCCs are associated with pro-inflammatory activity. As washing significantly reduced RCC immunomodulatory activity, implementation of this process may improve transfusion outcomes.