| To achieve long-term preservation of red blood cells, cryoprotectants are needed in the protective solution. However, some component in it may do harm to the body. So it should be removed before infusion to body. Up to now, routine method to remove protective agent in clinical practice is centrifugation (such as the automatic processing of red blood cells ACP215), which produce mechanical stress and stacking force leading to cell damage.Objective:To build a washing system, optimizing the washing conditions and, ultimately, wash freeze-thawing red blood cells, this research including three aspects. Firstly, in order to understand the change of glycerin concentration during washing process, the feasibility of osmotic pressure method was studied which reflect glycerol concentration of the frozen red blood cell by measuring osmotic pressure. Secondly, sharp change of osmotic pressure will cause serious damage of red blood cells, changes in erythrocyte volume and hemolysis under different osmotic is studied to optimize the washing condition of washing system. At last, this research will build a washing system based on the principle of dialysis and ultrafiltration,washing RBC which was frozen and thawed according to the optimal experiment scheme, and then the washing effect were evaluated.Methods:In order to obtain the correlation between osmotic pressure and the concentration of glycerol during washing process, different concentrations of glycerol erythrocytes salt solution were prepared with sodium chloride (NaCI), glycerol (C3H8O3) and whole blood to mimic the conditions of the washing process of frozen and thawed RBC. In addition, hemolysis test and repeatability were performed; Meanwhile, in order to obtain the relationship between osmotic pressure and red blood cell volume or hemolytic, Different gradient osmotic conditions were provided by impermeable materials, Sodium chloride; Hematocrit(Hct) were determined by the method of centrifugation after addition of whole blood. Cell morphology and diameter were recorded under the microscope. Freezing point osmotic pressure gauge was used to measure the osmotic pressure of supernatant. Cell volume were calculated on the basis of the data above. At last, washing RBC which was frozen and thawed according to the optimal experiment scheme by adjust the flow rates of washing liquid and blood, and then the washing effect were evaluated.Results:1、In hypotonic environment, the hemolysis rate were (87.3±4.3)%, (79.4±6.7)%, (68.5±6.6)%, (30.2±2.8)%, (6.6±1.0)%, (2.5±0.8)% respectively, mean corpuscular volume (MCV) were (184.1±32.7)fL, (216.4±62.9)fL, (185.2± 27.3)fL, (151.8±6.8)fL, (122.4±3.7)fL, (109.3±2.4)fL, when the osmotic pressure were 148 mOsm/kg,154 mOsm/kg,158 mOsm/kg,171mOsm/kg, 191mOsm/kg,236mOsm/kg; In hypertonic conditions, hemolysis rates were (2.1± 0.7)%, (2.5±0.6)%, (25.2±8.7)%, (55.8±13.8)%, MCV were (58.1±1.9)fL, (60.6 ±2.8)fL, (74.5±4.8)fL, (80.1±13.9)fL respectively, when the osmotic pressure were 810 mOsm/kg,1073 mOsm/kg,2460 mOsm/kg,3033 mOsm/kg; In isotonic environment, hemolysis rate was (1.9±0.8)%, MCV was (94.9±1.08)fL Those characteristic variation of red blood cell stored for one week have the same tendency as that for five weeks, but the degree of change had a slightly difference.2、When solute component contains only glycerol and NaCl, there is a good linear relationship between the concentration of glycerol and osmotic pressure, R2=0.9965. recovery rate were between 90%-110%, which is acceptable, coefficient of variation of repeatability and stability were less than 1%. With a high Recovery rate, There is a good linear relationship between the glycerol concentration and osmotic pressure when testing blood samples consisted of different glycerol concentration(y=15.253x +219.93, R2=0.9981). And coefficient variation of repeatability and stability are less than 2%. Interference experiments show that when the free hemoglobin is (1.71～ 15.16) g/L, blood osmotic pressure value are similar to glycerol concentrations. Osmotic pressure of the blood suspension is consistent with its supernatant’s, p= 0.156 the Wilcoxon signed-rank test show, the difference was not statistically significant.3、When the flow rate of washing liquid and blood are increased, curve slope increased which time is the horizontal axis, vertical axis is the osmotic pressure, the osmotic pressure of the solution can drop faster, washing efficiency is better. After washing by dialysis combined with ultrafiltration, red blood cell recovery of freezing-thawing RBC was (92.33±5.87)%, blood osmotic pressure was (291±1.77) mOsm/ kg, the concentration of glycerol was (0.24±0.12) g/L, the hemoglobin in supernatant was (0.74±0.13) g/L,these indexes can meet the national standard. Compared with ACP215,red blood cell recovery, glycerol of concentration, hemoglobin in supernatant were statistically significant, we found that the red blood cell recovery and glycerol of concentration were below ACP215, but hemoglobin in supernatant is higher than ACP215.conclusion:1、When osmotic pressure of solution is increasing, erythrocyte volume gradually reduce. In order to make the cell recovery rate> 80%, changes in red blood cell volume must be less than 1.5 times the isotonic osmotically active volume. In order to make the cell recovery rate>90%, changes in red blood cell volume must be less than 1.4 times the isotonic osmotically active volume.2、Due to the good linear relationship between the glycerol concentration and osmotic pressure, the concentration of glycerol can be calculated when osmolality is measured. This method is simple, fast, interference effect of Hemoglobin is small and less consumption of sample and reagent.3、It is a viable method to wash freeze-thawing red blood cells, the glycerol concentration and red blood cell count recovery are better than ACP215, but hemoglobin in supernatant is worse than ACP215. In addition, The experimental condition is not the best washing condition; which can be further improved, so as to further improve the recovery rate of red blood cells. |
PDF Full Text Request