Effect of trehalose-loading on red blood cell membrane / 中国实验血液学杂志

This study was purposed to evaluate the effect of trehalose-loading on physiological and biochemistry properties of red blood cell (RBC) membrane. The samples were divided into the control group (RBC without trehalose loading) and the test group (RBC with trehalose loading). Osmotic fragility reaction was used to determine the osmotic fragility change of loaded RBC membrane in NaCl solution of different osmotic concentration. Flow cytometry and deformeter were used to assay the integrality and deformability of the RBC, respectively. The results showed that the NaCl solution osmotic concentrations were 160 mOsm and 121.4 mOsm, respectively when the haemolysis rate was 50% of the control group and the test group. Flow cytometry data demonstrated that incubation of RBC in a hypertonic trehalose solution resulted in a fraction of cells with different complexity that attached to little Annexin V-FITC, and that it could be removed by washing and resuspending the RBC in an iso-osmotic (300 mOsm PBS) medium. The deformability of the loaded RBC descend, the statistical difference was significant between control and test groups (P < 0.01). It is concluded that the membrane physiological and biochemistry stability and membrane integrality of RBC in a hyper osmotic pressure can be retained after trehalose loading.

Optimization of lyophilization procedures for freeze-drying of human red blood cells / 南方医科大学学报

<p><b>OBJECTIVE</b>To investigate the different parameters of the lyophilization procedures that affect the recovery of the rehydrated red blood cells (RBCs).</p><p><b>METHODS</b>Human RBCs loaded in tubes were cooled with 4 different modes and subjected to water bath at 25 degrees celsius;. The morphological changes of the RBCs were observed to assess the degree of vitrification, and the specimens were placed in the freeze-dryer with the temperature set up at 40, -50, -60, -70 and -80 degrees celsius;. The rates of temperature rise of the main and secondary drying in the lyophilization procedures were compared, and the water residue in the specimens was determined.</p><p><b>RESULTS</b>The protectant did not show ice crystal in the course of freezing and thawing. No significant difference was found in the recovery rate of the rehydrated RBCs freeze-dried at the minimum temperature of -70 degrees celsius; and -80 degrees celsius; (P > 0.05). The E procedure resulted in the maximum recovery of the RBCs (83.14% ± 9.55%) and Hb (85.33% ± 11.42%), showing significant differences from the other groups(P < 0.01 or 0.05). The recovery of the RBCs showed a positive correlation to the water residue in the samples.</p><p><b>CONCLUSION</b>Fast cooling in liquid nitrogen and shelf precooling at -70 degrees celsius; with a moderate rate of temperature rise in lyophylization and a start dry temperature close to the shelf equilibrium temperature produce optimal freeze-drying result of human RBCs.</p>

Optimization of composition and concentration for lyophlizing protectant of human red blood cells / 中国实验血液学杂志

This study was purposed to investigate the effect of different compositions and concentrations of lyophilizing protectants on recovery of RBCs and hemoglobin (Hb) after rehydration of lyophilized RBCs. The RBC lyophilizing protectants composed of a series concentrations of PVP, trehalose and different osmotic protectants were applied for protecting lyophilizing process of RBCs, the recovery of RBCs and Hb after rehydration of lyophilized RBCs was detected. The results showed that there were significant differences in loss ratio of RBCs between protectants composed of different compositions and concentrations (p<0.05 or p<0.01). The loss ratio of RBCs in protectant containing 30% PVP40, 150 mmol/L trehalose and 2% BSA was minimum (0.02%), the loss ratio of RBCs in protectant containing 6% PVP 360, 100 mmol/L trehalose and 2% BSA was maximum (0.27%). The difference of effect between 150 and 50 mmol/L trehalose was statistically significant (p<0.01). The recovery rates of RBCs and Hb in protectants contained PVP40 of different concentrations were different after rehydration of lyophilized RBCs. The protectant containing 15% PVP40, 150 mmol/L trehalose and 2% BSA showed optimal protective efficacy for lyophilized RBCs, the recovery rates of RBCs and Hb were 61.29+/-4.11% and 62.49+/-5.91% respectively, which were statistically different from other protectants (p<0.01). The protectants containing glycerol displayed best efficiency in lyophilization too, the recovery rates of RBCs and Hb were 65.97+/-4.52% and 67.24+/-5.94%, respectively. It is concluded that the protectants composed of 0.8 mol/L glycerol, 15% PVP40, 150 mmol/L trehalose and 2% BSA (pH 7.3 ) may be used as the protectant lyophilizing human RBCs in future study.

Preliminary study on rehydrated conditions for lyophilized human red blood cells / 中国实验血液学杂志

The objective of this study was to investigate the effect of different rehydration conditions on recovery of the lyophilized red blood cells (RBC) so as to optimize the RBC rehydration. The different conditions, including different rehydration solution, the rehydration temperature, volume change rate of the lyophilized RBC rehydrated by the vapor firstly, were studied, the recovery rate and change of physiological and biochemical properties of the rehydrated RBC were detected. The results indicated that the solution of 10% (w/v) PVP40 in PBS showed the best effect, and the RBC recovery rate increased with increasing of rehydration temperature, and the optimal temperature of rehydration was at 37 degrees C. Pre-rehydration in condition of vapor could raise the RBC recovery rate, and promote the MCV and RDW to close to index of the fresh RBC, the deformability of the rehydrated RBC was no serious as compared with RBC preserved in conventional condition, but the activity level of ATP, G-6-PD, SOD, 2, 3-DPG of the rehydrated RBC less decreased. It is concluded that the optimal rehydration conditions for lyophilized RBC are pre-rehydration in the 37 degrees C with vapor firstly, PBS + 10% (w/v) PVP40 rehydration solution and rehydration temperature at 37 degrees C, but the protection of RBC membrane needs to be furtherly studied.

Optimization of trehalose loading in red blood cells before freeze-drying / 中国实验血液学杂志

The key points for better protection of trehalose in freeze-drying red blood cells (RBCs) are to resolve non-osmosis of trehalose to red blood cells and to make cytoplasmic trehalose to reach effective concentration. This study was aimed to investigate the regularity of loading RBCs with trehalose, screen out optimal loading condition and evaluate the effect of trehalose on physico-chemical parameters of RBCs during the period of loading. The cytoplasmic trehalose concentration in red blood cells, free hemoglobin and ATP level were determined at different incubation temperatures (4, 22 and 37 degrees C), different trehaolse concentrations (0, 200, 400, 600, 800 and 1000 mmol/L) and different incubation times (2, 4, 6, 8 and 10 hours), the cytoplasmic trehalose, free hemoglobin (FHb), hemoglobin (Hb) and mean corpuscular volume (MCV) in fresh RBCs and RBCs stored for 72 hours at 4 degrees C were compared, when loading condition was ensured. The results showed that with increase of incubation temperature, time and extracellular trehalose concentration, the loading of trehalose in RBCs also increased. Under the optimal loading condition, cytoplasmic trehalose concentration and free hemoglobin level of fresh RBCs and RBCs stored for 72 hours at 4 degrees C were 65.505 +/- 6.314 mmol/L, 66.2 +/- 5.002 mmol/L and 6.567 +/- 2.568 g/L, 16.168 +/- 3.922 g/L respectively. It is concluded that the most optimal condition of loading trehalose is that fresh RBCs incubate in 800 mmol/L trehalose solution for 8 hours at 37 degrees C. This condition can result in a efficient cytoplasmic trehalose concentration. The study provides an important basis for long-term preservation of RBCs.

Aggregation after rehydration of lyophilized platelets / 中国实验血液学杂志

This study was aimed to investigate the aggregation of rehydrated-lyophilized platelets. The aggregation rate of fresh and rehydrated-lyophilized platelets were measured by using thrombin, ristocetin, ADP and collagen as inductors and APACT2 aggregameter; the effects of intra- and extra-cellular trehalose on maximum aggregation rate of rehydrated-lyophilized platelets were detected by using ADP as an inductor. The results showed that the aggregation rate of fresh platelets was all about 100%, while aggregation rate of rehydrated lyophilized platelets was (70.17 +/- 7.36)%, (15.3 +/- 2.81)%, (68.67 +/- 6.86)%, (64.67 +/- 11.6)% respectively, when the concentration of thrombin, ristocetin, ADP and collagen was 1 U/ml, 1.6 mg/ml, 20 micromol/L and 2 microg/ml. The maximum aggregation rates of rehydrated-lyophilized platelets in intra- and extra-cellular trehalose, extracellular trehalose and blank control groups were (66.0 +/- 4.69)%, (25.3 +/- 2.42)% and (11.5 +/- 1.87)% (P < 0.01), meanwhile there was significant difference of rehydrated-lyophilized platelet aggregation rate between intra- and extra-cellular trehalose and extracellular trehalose groups (P < 0.01). It is concluded that the concentrations of thrombin (1 U/ml), ristocetin (1.6 mg/ml), ADP (20 micromol/L) and collagen (2 microg/ml) are optimal for platelets aggregation tests, the internal and extracellular trehalose significantly enhance the aggregation of rehydrated-lyophilized platelets.