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.

Multicenter evaluation of the IL-3-pSTAT5 assay to assess the potency of cryopreserved stem cells from cord blood units: The BEST Collaborative study.

BACKGROUND: The IL-3-pSTAT5 assay, a new, rapid, and standardized flow-cytometry-based assay may compensate for several limitations of the colony-forming unit (CFU) assay typically used for stem cell potency assessments of cord blood units (CBU). We performed an inter-laboratory evaluation of the performance of this new assay. STUDY DESIGN AND METHODS: This Biomedical Excellence for Safer Transfusion (BEST) Collaborative multicenter, international study included 15 participants from public cord blood banks (CBBs), CBB-supporting research laboratories, and stem cell laboratories. To perform the IL-3-pSTAT5 assay, participating centers received reagents, instructions, and 10 blind CBU samples, including eight normal samples and two samples exposed to a transient warming event. We measured inter-laboratory agreement qualitatively (proportion of correctly classified samples) and quantitatively (coefficient of variation [CV], correlation coefficients, receiver operating characteristics (ROC) curve, and intraclass correlation coefficient [ICC]). RESULTS: The qualitative agreement was 97.3% (i.e., 107/110; Fleiss' kappa = 0.835). The average CV on a per-sample basis was 11.57% among all samples, 8.99% among normal samples, and on a per-center basis was 9.42% among normal samples. In a correlation matrix that compared results across centers, the mean Pearson's correlation coefficient was 0.88 (standard deviation = 0.04). The ICC was 0.83 (95% confidence interval = 0.68-0.95). The area under the curve (AUC) from the ROC curve was 0.9974. DISCUSSION: Excellent qualitative and quantitative agreement was exhibited across laboratories. The IL-3-pSTAT5 assay may therefore be implemented in flow cytometry laboratories to rapidly and reliably provide standardized measures of stem cell potency in CBUs.

Evaluation of the functional properties of cryopreserved buffy coat-derived monocytes for monocyte monolayer assay.

BACKGROUND: Monocyte monolayer assay (MMA) is a compatibility testing method for evaluating the clinical significance of red blood cell (RBC) alloantibodies. Time-consuming monocyte isolation procedures and requirement for fresh monocytes have limited application of the MMA. The aim of this study was to develop and assess the utility and efficacy of cryopreserved buffy coat (BC)-derived monocytes for MMA application. STUDY DESIGN AND METHODS: Peripheral blood mononuclear cells (PBMNCs) were isolated from BC or peripheral blood (PB) and pooled and BC PBMNCs were cryopreserved. Monocytes from pooled PBMNCs were incubated with anti-D-sensitized, anti-Scianna2 (Sc2)-sensitized, anti-AnWj-sensitized, or anti-Jra -sensitized RBCs or lipopolysaccharide (LPS). MMA phagocytic index (PI) and membrane integrity were determined microscopically, and cytokine release was measured by Luminex technology. RESULTS: PBMNC isolation rates from fresh BC and PB were not comparable (67.4 ± 6.3 and 75.8 ± 7.7% respectively, p = 0.024). There was no significant difference in PBMNC membrane integrity (fresh PB, 100%; fresh BC, 100%; cryopreserved BC, 95.2 ± 1.2%), postwash recovery (fresh PB, 85.9 ± 3.1; fresh BC, 86.9 ± 6.7; cryopreserved BC, 84.8 ± 5.1), or monocyte PI (fresh PB, 82 ± 10; fresh BC, 77 ± 11; cryopreserved BC = 80 ± 6). Monocytes from pooled cryopreserved BC PBMNCs reacted with RBCs sensitized with anti-D and RBC alloantibodies, including anti-Sc2, anti-Jra , and anti-AnWj. CONCLUSIONS: Monocytes from pooled cryopreserved BC PBMNCs can be used reliably to evaluate phagocytic responses of sensitized RBCs and to assess clinical significance of RBC alloantibodies.

The immune-stimulation capacity of liposome-treated red blood cells.

Our in vivo studies on a rat model established safety of transfusing liposome-treated red blood cells (RBCs) but identified the potential for immune modulation as related to transfusion efficacy of liposome-treated RBCs. The aim of this study was at assessing the impact of liposome-induced membrane changes on the immune profile of liposome-treated RBCs by (a) evaluating their interaction with endothelial cells and monocytes; and (b) the resulting immune response derived from this interaction, in the form of cytokine release, adhesion molecules expression and phagocytosis. Unilamellar liposomes were synthesized to contain unsaturated phospholipids (1,2-dioleoyl-sn-glycero-3-phosphocholine [DOPC]:CHOL, 7:3 mol%). The human RBCs immune profile was assessed by incubating control and DOPC-treated RBCs with human umbilical vein endothelial cells (HUVECs) and monocytes. Cytokine release measured by Luminex technology, vascular cell adhesion molecule (VCAM)-1 and E-selectin on HUVECs measured by flow cytometry, and the erythrophagocytic activity of monocytes by monocyte monolayer assay (MMA) were determined. Fibroblast growth factor [FGF]-2 was the only cytokine released by HUVECs that remained increased after incubation with DOPC-treated RBCs compared to control throughout storage. The expression of both VCAM-1 (15.3 ± 5.6% versus 6.3 ± 0.9%, p = 0.008) and E-selectin (18.0 ± 6.3% versus 6.6 ± 0.7%, p = 0.004) by HUVECs were significantly increased after incubation with DOPC-treated RBCs at day 2 of storage. The MMA resulted in phagocytic indexes of zero for both control and DOPC-treated RBCs at day 2 and 42 of storage. The liposome treatment did not result in significant changes to the immune profile of stored DOPC-treated RBCs. These findings combined with previous in vivo results, make liposome treatment a potential candidate for application in RBC preservation and open the possibility for clinical use with other cell types.

Characteristics of Extracellular Vesicles in Red Blood Concentrates Change with Storage Time and Blood Manufacturing Method.

BACKGROUND: Extracellular vesicles (EVs) in blood products are potential effectors of inflammation and coagulation after transfusion. The aim of this study was to assess the impact of different blood manufacturing methods and duration of hypothermic storage on the EV subpopulations in relation to other in vitro quality parameters of red blood cell concentrate (RCC) products. METHODS: RCCs were produced using whole blood filtration (WBF) or red cell filtration (RCF) (n = 12/method), refrigerated for 43 days, and evaluated for EV size profile and concentration, red cell deformability, ATP and 2,3-DPG, hemolysis, and hematological indices. RESULTS: The total number of EVs increased significantly with storage in both methods, and WBF-RCCs contained the higher numbers of EVs compared to RCF-RCCs. The concentration of small EVs was greater in WBF-RCCs versus RCF-RCCs, with difference between the two methods observed on day 43 of storage (p = 0.001). Throughout storage, significant decreases were identified in ATP, 2,3-DPG, and EImax, while an increase in hemolysis was observed in both RCC products. CONCLUSION: The dynamic shift in the size and concentration of the EV subpopulations is dependent on the blood manufacturing method and length of storage. Better understanding of the potential clinical implications of these heterogeneous populations of EVs are needed.

Extracellular vesicle characteristics in stored red blood cell concentrates are influenced by the method of detection.

Extracellular vesicles (EVs), including microvesicles and exosomes, are small phospholipid vesicles (≤1µm in diameter) that are present in blood products, accumulate during storage, and have a potential transfusion-related immunomodulatory role. Knowledge of EVs in stored blood products is limited due to the challenges and difficulties in detecting these heterogeneous submicron-sized vesicles. The aim of this study was to assess the impact of different approaches to characterize EVs in stored RBC products. Quantification and size-profiling of EVs in leukoreduced red cell concentrates (RCCs) were examined on day 3, 7, 21, and 42 of storage using tunable resistive plus sensing (TRPS), flow cytometer (FC), and dynamic light scatting (DLS) methods. Using the TRPS method, the concentration of EVs<200nm significantly increased throughout storage (p<0.05). This change in exosome concentration was not detectable with FC or DLS due to limitations in their ability to resolve particles <200nm and/or accurately determine EV concentration. Both the TRPS and FC demonstrate that the concentration of EVs≥200nm significantly increases in RCCs by day 42/43 compared to EVs present on day 3 (p<0.001). As the DLS measures the average size of particles in suspension, only an increase in the zeta-average size was observed during storage. EV size and concentration in RBC products is significantly influenced by the length of storage. Overall, this study shows that combining technologies may be important to improve the characterization and study of EVs in stored RCCs.

Effect of liposome-treated red blood cells in an anemic rat model.

CONTEXT: Liposomes have been shown to improve human red blood cell (RBC) in vitro quality by minimizing membrane damage occurring during 42-d hypothermic storage. Small animal models are necessary to evaluate novel blood products and guide future clinical studies. OBJECTIVES: The aim of this study was to assess the effect of liposome treatments on rat RBC hypothermic storage lesion (HSL) and to examine in vivo outcomes of transfusing liposome treated RBCs in a rat model. MATERIALS AND METHODS: Unilamellar liposomes were synthesized which contained saturated (DPPC:CHOL, 7:3 mol%), unsaturated (DOPC:CHOL, 7:3 mol%), saturated charged (DPPC:CHOL:PS, 6:3:1 mol%), and unsaturated charged (DOPC:CHOL:PS, 6:3:1 mol%) phospholipids. After liposome treatment, rat RBC quality was assessed by percent hemolysis, deformability, aggregation, hematological indices, microvesiculation, and cholesterol/phospholipid concentrations. An anemic rat model of myocardial ischemia and reperfusion (I/R) was used to evaluate the outcomes of transfusing liposome-treated RBCs. RESULTS: All four liposome treatments resulted in significant decreases in hemolysis, with the most prominent effect seen with DOPC-liposomes (DOPC: 1.6 ± 0.1% versus control: 3.1 ± 0.2%, p = 0.015). RBCs treated with uncharged liposomes had lower hemolysis compared with charged liposomes (3.4 ± 0.2% versus 3.9 ± 0.4%, p = 0.010). The in vivo study showed no significant difference in the hemoglobin levels and infarct size (53.3 ± 13.1% versus 45.3 ± 8.4%, p = 0.223) between liposome and control groups. DISCUSSION AND CONCLUSION: Liposome treatment improved in vitro quality of stored rat RBCs. However, the changes observed in vitro were not sufficient to improve the in vivo outcomes of myocardial I/R in anemic rats transfused with liposome-treated RBCs.

Cryopreserved amniotic membrane as transplant allograft: viability and post-transplant outcome.

Amniotic membrane (AM) transplantation is increasingly used in ophthalmological and dermatological surgeries to promote re-epithelialization and wound healing. Biologically active cells in the epithelial and stromal layers deliver growth factors and cytokines with anti-inflammatory, anti-bacterial, anti-immunogenic and anti-fibrotic properties. In this work, confocal microscopy was used to show that our cryopreservation protocol for AM yielded viable cells in both the stromal and epithelial layers with favorable post-transplant outcome. AM was obtained from Caesarean-section placenta, processed into allograft pieces of different sizes (3 cm × 3 cm, 5 cm × 5 cm, and 10 cm × 10 cm) and cryopreserved in 10 % dimethyl sulfoxide using non-linear controlled rate freezing. Post-thaw cell viability in the entire piece of AM and in the stromal and epithelial cell layers was assessed using a dual fluorescent nuclear dye and compared to hypothermically stored AM, while surveys from surgical end-users provided information on post-transplant patient outcomes. There was no significant statistical difference in the cell viability in the entire piece, epithelial and stromal layers regardless of the size of allograft piece (p = 0.092, 0.188 and 0.581, respectively), and in the entire piece and stromal layer of hypothermically stored versus cryopreserved AM (p = 0.054 and 0.646, respectively). Surgical end-user feedback (n = 49) indicated that 16.3 % of AM allografts were excellent and 61.2 % were satisfactory. These results support the expanded clinical use of different sizes of cryopreserved AM allografts and address the issue of orientation of the AM during transplant for the treatment of dermatological defects and ocular surface disorders.

Differences in Rat and Human Erythrocytes Following Blood Component Manufacturing: The Effect of Additive Solutions.

BACKGROUND: Small animal models have been previously used in transfusion medicine studies to evaluate the safety of blood transfusion products. Although there are multiple studies on the effects of blood banking practices on human red blood cells (RBCs), little is known about the effect of blood component manufacturing on the quality of rat RBCs. METHODS: Blood from Sprague-Dawley rats and human volunteers (n = 6) was collected in CPD anticoagulant, resuspended in SAGM or AS3, and leukoreduced. In vitro quality was analyzed, including deformability, aggregation, microvesiculation, phosphatidylserine (PS) expression, percent hemolysis, ATP, 2,3-DPG, osmotic fragility, and potassium concentrations. RESULTS: Compared to human RBCs, rat RBCs had decreased deformability, membrane rigidity, aggregability, and microvesiculation after component manufacturing process. Rat RBCs in SAGM showed higher hemolysis compared to human RBCs in SAGM (rat 4.70 ± 0.83% vs. human 0.34 ± 0.07%; p = 0.002). Rat RBCs in AS3 had greater deformability and rigidity than in SAGM. The number of microparticles/µl and the percentage PS expression were lower in rat RBCs in AS3 than in rat RBCs in SAGM. Hemolysis was also significantly lower in AS3 compared to SAGM (2.21 ± 0.68% vs. 0.87 ± 0.39%; p = 0.028). CONCLUSION: Rat RBCs significantly differ from human RBCs in metabolic and membrane-related aspects. SAGM, which is commonly used for human RBC banking, causes high hemolysis and is not compatible with rat RBCs.

Phospholipidomics reveals differences in glycerophosphoserine profiles of hypothermically stored red blood cells and microvesicles.

During their normal in vivo life cycle erythrocytes (red blood cells, RBCs) undergo biochemical changes leading to membrane microvesiculation and shedding. RBC microvesiculation also occurs in vitro under conditions of blood bank storage, so microvesicles (MVs) accumulate in the storage (preservation) medium over storage time. Considerable effort has been put into gaining a mechanistic understanding of the RBC microvesiculation process, as this is crucial to better understand RBC biology in disease and in health. Additionally, MVs accumulated in stored RBCs have been implicated in transfusion adverse inflammatory reactions, with chloroform extractable compounds, thus lipophilic, known to trigger the effect. However, because thin layer chromatography resolution of RBC and MV lipids has always enabled one to conclude high compositional similarities, in depth analysis of MV lipids has not been extensively pursued. Here we present an orbitrap mass spectrometry (MS) approach to compare the phospholipid composition of RBCs and MVs from leukoreduced, hypothermically (2-6°C) stored RBC units. We used shotgun MS analysis and electrospray ionization (ESI) intra-source separation, and demonstrated high similarity of compositional profiles, except for glycerophosphoserines (PS). Contrasting abundances of PS 38:4 and PS 38:1 characterized MV and RBC profiles and suggested that storage-associated microvesiculation possibly involves shedding of specific membrane rafts. This finding indicates that phospholipidomics could likely contribute to a better understanding of the RBC microvesiculation process.

The effects of rejuvenation during hypothermic storage on red blood cell membrane remodeling.

BACKGROUND: Our previous studies showed that hypothermic storage (HS) induces red blood cell (RBC) microparticle (RMP) generation and changes in phosphatidylserine (PS) and CD47 expression on RBCs and RMPs. The aim of this study was to evaluate the effect of cold rejuvenation treatment at multiple time points during storage on these prehemolytic indicators of RBC membrane storage lesion. STUDY DESIGN AND METHODS: Leukoreduced RBC units in saline-adenine-glucose-mannitol were used to generate three groups: untreated controls, sham-treated units, and units treated with a cold (1-6°C) rejuvenation solution on Day 28, 35, or 42 of HS. Units were assessed for hemolysis, adenosine triphosphate (ATP) concentration, lipid composition, and RMP generation, as well as PS and CD47 expression throughout 49 days of HS. RESULTS: Rejuvenation treatment led to a significant increase in ATP concentration in all units, irrespective of treatment day. There were no significant differences between sham- and rejuvenation-treated RBC samples in the levels of PS externalization, CD47 expression, or the rate of RMP formation. RBCs rejuvenated on Day 28 were enriched in glycerophosphocholine (+23.5%), depleted in sphingomyelin (-14%), and slightly depleted in cholesterol (-3.5%). CONCLUSION: Cold rejuvenation in hypothermically stored RBCs affects the lipid composition of RBCs and respective RMPs in a time-dependent fashion.

Optimizing Access to Unrelated Donors in Canada: Re-Examining the Importance of Donor Factors on Outcomes Following Hematopoietic Cell Transplantation.

HLA-matched allogeneic hematopoietic cell transplantation (HCT) is a curative therapy for many patients. Unrelated HLA-matched donors are the most frequently used donor for HCT. When more than one donor transplant option is available, transplant centers can select donors based on non-HLA factors. With improved ability to prevent and treat immune complications, such as graft-versus-host disease and infections, it may be possible to proceed more often using HLA-mismatched donors, allowing greater consideration of non-HLA factors, such as donor age, CMV serostatus, and ABO blood group matching, which have demonstrated important impacts on transplant outcomes. Additional factors to consider are donor availability rates and the usage of domestic donors to optimize outcomes. A review of non-HLA factors and considerations on the selection of optimal unrelated donors for HCT are provided within this updated current context.

Storage of red blood cells affects membrane composition, microvesiculation, and in vitro quality.

BACKGROUND: During storage detrimental biochemical and biomechanical changes occur within a red blood cell (RBC). RBC microparticles (RMPs) produced during storage have been identified as biomarkers of RBC quality, being potentially immunogenic and inhibitory to nitric oxide regulation. STUDY DESIGN AND METHODS: In this study, microvesiculation and changes in the composition of the RBC membrane were investigated throughout 49 days of storage and were correlated with in vitro assays examining membrane quality. Leukoreduced RBC units produced using the buffy coat method were collected and stored at 1 to 6°C and were tested weekly for hemolysis, osmotic fragility, deformability, ATP, hematologic indices, and morphology. Microvesiculation was assessed using multicolor flow cytometry. High-performance liquid chromatography and mass spectrometry were used to determine the composition and quantity of phospholipids (PLs) and cholesterol (C) on Days 2 and 43. RESULTS: The assessment of RBCs throughout storage revealed significant increases in percent hemolysis, while significant decreases in ATP concentrations, and the mean corpuscular hemoglobin concentration were observed. Flow cytometry analysis revealed a significant increase in the mean number of microparticles per microliter during storage. Throughout storage, significant decreases were identified in the amount of PLs and total lipids within the RBC membrane. No significant change in the amount of C in the RBC membrane was identified. CONCLUSION: Significant changes to the RBC membrane occur during storage. The length of storage will influence RMP generation, osmotic fragility, hemolysis, and changes in deformability. These changes in RBC in vitro quality may contribute to transfusion reactions and negative posttransfusion outcomes.

Rejuvenation of ATP during storage does not reverse effects of the hypothermic storage lesion.

BACKGROUND: Hypothermic storage (HS) of red blood cells (RBCs) leads to a progressive deterioration of cell quality. Mitigating these deleterious changes could allow maintenance or even an improvement of RBC in vitro quality. The aim was to determine the effect of a cold rejuvenation treatment of RBCs and in particular to assess the connection between ATP levels, RBC deformability, and morphology during RBC storage. STUDY DESIGN AND METHODS: A pool-and-split design of leukoreduced CPD-saline-adenine-glucose-mannitol-packed RBC units was used to generate three groups: untreated controls, sham-treated units, and units treated with a cold (1-6 °C) rejuvenation solution on Day 28, 35, or 42 of cold storage. Units were followed until Day 49 of storage and assessed for ATP concentration, morphology, deformability, and other in vitro quality variables including hemolysis, pH, and supernatant potassium levels. RESULTS: At every treatment time, rejuvenation was associated with a significant increase in intracellular ATP (p < 0.01). On Day 28, rejuvenation was accompanied by a significant decrease in deformability 1 week after treatment (p < 0.01). Rejuvenation on Day 28, but not Day 35 or 42, was also associated with a significant change in morphology (p < 0.01). Of the in vitro quality variables measured, most changed during cold storage, but differences among treatment groups were not observed. CONCLUSION: The results of our study demonstrate a cold rejuvenation of RBCs during HS increases intracellular ATP, but that this change does not ameliorate, or exacerbate, the metabolic or biochemical symptoms of the storage lesion.

Liposomes alter thermal phase behavior and composition of red blood cell membranes.

Unilamellar liposomes composed of natural phospholipids provide a new promising class of protective agents for hypothermic storage, cryopreservation, or freeze-drying of red blood cells (RBCs). In this study, FTIR spectroscopy, MALDI-TOF MS, and colorimetric assays were used to investigate the effects of liposomes composed of a homologous series of linear saturated phosphatidylcholine phospholipids (18:0; 16:0; 14:0; 12:0) on RBC membranes. RBCs were incubated with liposomes at 37°C and both the liposomal and the RBC fraction were analyzed after incubation. FTIR studies showed that liposomes composed of short acyl chain length lipids cause an increase in RBC membrane conformational disorder at suprazero temperatures, whereas long acyl chain length lipids were found to have little effects. The increased lipid conformational disorder in the RBC membranes coincided with a decrease in the cholesterol-to-phospholipid ratio. The opposite effects were found in the liposomes after incubation with RBCs. MALDI-TOF MS analysis showed the presence of short acyl chain length lipids (14:0 and 12:0) in RBC membranes after incubation, which was not observed after incubation with liposomes containing long acyl chain length lipids (18:0 and 16:0). Liposomes alter RBC membrane properties by cholesterol depletion and lipid addition.

A Bayesian adjustment for multiplicative measurement errors for a calibration problem with application to a stem cell study.

We develop a Bayesian approach to a calibration problem with one interested covariate subject to multiplicative measurement errors. Our work is motivated by a stem cell study with the objective of establishing the recommended minimum doses for stem cell engraftment after a blood transplant. When determining a safe stem cell dose based on the prefreeze samples, the postcryopreservation recovery rate enters in the model as a multiplicative measurement error term, as shown in the model. We examine the impact of ignoring measurement errors in terms of asymptotic bias in the regression coefficient. According to the general structure of data available in practice, we propose a two-stage Bayesian method to perform model estimation via R2WinBUGS (Sturtz, Ligges, and Gelman, 2005, Journal of Statistical Software 12, 1-16). We illustrate this method by the aforementioned motivating example. The results of this study allow routine peripheral blood stem cell processing laboratories to establish recommended minimum stem cell doses for transplant and develop a systematic approach for further deciding whether the postthaw analysis is warranted.

Liposomes composed of unsaturated lipids for membrane modification of human erythrocytes.

Previous studies have shown that certain saturated lipids protect red blood cells (RBCs) during hypothermic storage but provide little protection during freezing or freeze-drying, whereas various unsaturated lipids destabilize RBCs during hypothermic storage but protect during freezing and freeze-drying. The protective effect of liposomes has been attributed to membrane modifications. We have previously shown that cholesterol exchange and lipid transfer between liposomes composed of saturated lipids and RBCs critically depends on the length of the lipid acyl chains. In this study the effect of unsaturated lipids with differences in their number of unsaturated bonds (18:0/18:1, 18:1/18:1, 18:2/18:2) on RBC membrane properties has been studied. RBCs were incubated in the presence of liposomes and both the liposomal and RBC fraction were analyzed by Fourier transform infrared spectroscopy (FTIR) after incubation. The liposomes caused an increase in RBC membrane conformational disorder at suprazero temperatures. The fluidizing effect of the liposomes on the RBC membranes, however, was found to be similar for the different lipids irrespective of their unsaturation level. The gel to liquid crystalline phase transition temperature of the liposomes increased after incubation with RBCs. RBC membrane fluidity increased linearly during the first 8 hours of incubation in the presence of liposomes. The increase in RBC membrane fluidity was found to be temperature dependent and displayed Arrhenius behaviour between 20 and 40°C, with an activation energy of 88 kJ mol⁻¹. Taken together, liposomes composed of unsaturated lipids increase RBC membrane conformational disorder, which could explain their cryoprotective action.

Emerging Role for Use of Liposomes in the Biopreservation of Red Blood Cells.

SUMMARY: Biopreservation is the process of maintaining the integrity and functionality of cells held outside the native environment for extended storage times. The development of red blood cell (RBC) biopreservation techniques that maintain in vitro RBC viability and function represents the foundation of modern blood banking. The biopreservation of RBCs for clinical use can be categorized based on the techniques used to achieve biologic stability, including hypothermic storage and cryopreservation. This review will examine the emerging role of liposomes in the RBC biopreservation, including the incorporation of liposomes into RBC membranes as an effective approach for minimizing RBC hypothermic storage membrane lesion and use of liposomes as a permeabilization strategy for the intracellular accumulation of novel intracellular cryoprotectants. Integration of current biopreservation research with blood banking practices offers enormous potential for future improvements of safety and efficacy of RBC transfusion.

Effects of trehalose-loaded liposomes on red blood cell response to freezing and post-thaw membrane quality.

We are investigating the use of liposomes, which are synthetic, microscopic vesicles, for the intracellular delivery of trehalose into mammalian cells. This study focuses on the effects trehalose-containing liposomes improve the recovery and membrane quality of human RBCs following cryopreservation. Unilamellar liposomes consisting of a lipid bilayer composed of DPPC, PS and cholesterol (60:30:10 mol%) were synthesized using an extrusion method. Liposome-treated RBCs (l-RBCs) were resuspended in either physiological saline, 0.3M trehalose or liposome solution, then cooled with slow (0.95+/-0.02 degrees C/min), medium (73+/-3 degrees C/min) and fast (265+/-12 degrees C/min) cooling rates and storage in liquid nitrogen, followed by a 37 degrees C thawing step. RBC post-thaw quality was assessed using percent recovery, RBC morphology, PS and CD47 expression. Liposome treatment did not adversely affect the RBC membrane. Post-thaw recovery of l-RBCs was significantly higher (66%+/-5% vs 29%+/-4%) compared to control RBCs (c-RBC, p=0.003). Medium and high cooling rates resulted in significantly higher cell recovery compared to a slow cooling rate (p=0.039 and p=0.041, respectively). The recovery of l-RBCs frozen in liposome solution and trehalose solution was significantly higher than that of l-RBCs frozen in NaCl solution for all three cooling rates (p=0.021). Flow cytometry and morphology assessment showed that liposome treatment resulted in improved post-thaw membrane quality. There was no statistically significant difference in the post-thaw recovery between RBCs treated with liposomes containing trehalose in their aqueous core and RBCs treated with liposomes containing saline in their aqueous core (p=0.114). Liposome treatment significantly improves the recovery and membrane integrity of RBCs following low temperature exposure.

Effect of Liposome Treatment on Hemorheology and Metabolic Profile of Human Red Blood Cells During Hypothermic Storage.

BACKGROUND: Ex vivo cold storage of red blood cells (RBCs) for transfusion has long been associated with hypothermic storage lesions. It has been proposed that liposomes can be used to mitigate hemorheological elements of RBC membrane storage lesions. This study aimed to determine the appropriate liposome treatment time and assess the effects of liposome treatment on RBC's hemorheological and metabolic profiles. MATERIALS AND METHODS: Unilamellar liposomes were synthesized to contain a bilayer of 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC):cholesterol (7:3 mol%). Packed human RBCs (n = 4) were divided into untreated control (HEPES-NaCl solution) and liposome-treated samples (2 mM DOPC liposomes) and treated at days 2, 21, and 42 of hypothermic storage. RBC quality assessment included percent hemolysis, deformability, aggregation, hematological indices, microvesiculation, supernatant potassium, adenosine triphosphate (ATP), and 2,3-diphosphoglycerate (2,3-DPG). RESULTS: Among the parameters affected by liposome treatment time were deformability, aggregation amplitude (Amp), mean corpuscular hemoglobin, mean corpuscular hemoglobin concentration, and microparticle mean fluorescence intensity. After 6 weeks of storage, aggregation index (AI) and Amp were significantly increased in liposome-treated RBCs (AI: 45.38 ± 1.92% vs. 41.54 ± 4.10%, p = 0.020; Amp: 16.38 ± 2.17 arbitrary units [au] vs. 12.22 ± 3.29 au, p = 0.019). Despite comparable hemolysis levels at 3 and 6 weeks, DOPC-treated RBCs showed significantly increased potassium levels for the same time points (3 weeks: 31.2 ± 2.7 mmol/L vs. 30.8 ± 2.7 mmol/L, p = 0.007; 6 weeks: 45.0 ± 3.0 mmol/L vs. 43.8 ± 3.4 mmol/L, p = 0.013). ATP and 2,3-DPG levels were comparable throughout storage. CONCLUSIONS: Liposome treatment seemed to be more beneficial when performed at the beginning of storage up to day 21. DOPC liposome treatment resulted in an improvement in human RBC hemorheology upon storage, with no significant impact on metabolic profile.