Study On The Measurement,distribution And Influences For Metabolism Of Lyophilized Red Blood Cells' Residual Water

Objective In order to investigate the best method to mensurate the lyophilized red blood cell's residual water,we explore the effects of lyophilized condition on red blood cell's residual water such as temperature,vacuity,drying time;Meanwhile,we also explored the effects of the distribution of residual water and the metabolism of red blood cells on the basis of previous research foundation.The experiment also provide experimental and theoretical basis for further study on the influence of residual water on the cryopreservation of red blood cells.Methods For the lyophilized red blood cells prepared by the same procedure,we use Karl-Fisher titration,thermogravimetric analysis and nuclear magnetic resonance(NMR)separately to mensurate the residual water,then analyze the differences.Use the same procedure to lyophilize equivalent blood ghost contained protectant,RBCs contained protectant,blood ghost without protectant and RBCs without protectant,mensurate the residual water separately and then analyze the results.Controlling the procedure including temperature,time,and vacuity in order to prepare lyophilized RBCs contained different residual water,using Karl-Fisher titration to determine the residual water and conserving at room temperature.Mensurating the ATP level,the SOD activity and the recovery rate of lyophilized cells(before lyophilization,one week,one month,three months and six months after lyophilization)after rehydration washing separately.Finally,analyzing the variation and association in each groups.Results1,The study use three different methods to determine the residual water content of lyophilized RBCs,lyophilized blood ghost and water standard.The protectant contained12%(w/v)glycerin concentration.The residual water of lyophilized RBCs contained protectant RBCs without protectant determined and water standard by Karl-Fisher titration was(3.37±0.05)%,(1.22±0.09)%,(5.14±0.13)%;The residual water of lyophilized RBCs contained protectant RBCs without protectant determined and water standard by thermogravimetric analysis was(12.40±0.56)%,(4.60±0.78)%,(5.28±0.16)%;The residual water of lyophilized RBCs contained protectant,RBCs without protectant and water standard determined by the method of NMR was(0.78±0.09)%,(0.89±0.12)%,(4.99±0.18)%;The difference between the three groups was statistically significant(P<0.05)except the group of water standard.Based on the LSD test there was no statistical significance(P > 0.05)in the group of lyophilized RBCs and lyophilized blood ghost.The residual water cantent between blood ghost and RBCs was no difference.The Karl Fisher titration could determinate the residual water content of lyophilized RBCs with or without protectant accurately,while the thermogravimetric analysis could be used for the lyophilized RBCs without glycerol,the method of NMR is relatively unreliable.2,Six groups of lyophilized RBCs prepared through the control of lyophilized condition contained different residual water was 2.80%,5.12%,9.10%,10.77%,11.73%,12.45% respectively.Rehydration washing at one week,one month,three months and six months after lyophilize.Compared with RBCs before lyophilize,the ATP level and the SOD activity of each group were decreased with time,the cellular functions tend to be gentle as time in the group of residual water content between 2.80% and 5.12%.Conclusion1,Fisher method,thermogravimetric method and the method of NMR could all be used to measure the residual water content,there were significant differences betweenthe determination of the absolute value.The measured results of thermogravimetric analysis are on the high side,The measured results of the method of NMR are pretty low.2,The residual water of lyophilized RBCs and lyophilized blood ghost was no statistical significance,showed that the residual water of lyophilized RBCs was distributed on the membrane of RBCs.3,The cellular functions tend to be gentle as time in the group of residual water content between 2.80% and 5.12% which suggest to control the residual water content in the interval will effectively increase the red blood cell lyophilized preservation time.