Study On A Multi-parameter Evaluation System For The Oxygen Supply Efficiency Of Storage Red Blood Cells

Hemoglobin in red blood cells is the main carrier of oxygen transport in the body,and blood transfusion is an important means of clinical treatment.Infusion and storage of red blood cells are mainly used in the treatment of all kinds of blood loss and anemia correction,in order to maintain or restore the transport of oxygen in the body.The current infusion standard of stored red blood cells is mainly based on the number of red blood cells and hemoglobin,and less attention is paid to the functional quality of stored red blood cells.The researchers took hemoglobin oxygen affinity as the main factor affecting the change of oxygen supply efficiency of stored red blood cells,and the partial pressure of oxygen at half saturation(P₅₀),the main evaluation index of hemoglobin oxygen affinity,was used to evaluate the oxygen supply efficiency of stored red blood cells.In different studies,it was found that the P₅₀ value of stored red blood cells decreased significantly during the storage period,but the storage period did not directly correspond to the effect of oxygen supply after erythrocyte infusion.The above phenomena indicate that the oxygen affinity of hemoglobin is not the only factor affecting the oxygen supply efficiency of stored red blood cells,and a single P₅₀ value is not enough to evaluate the oxygen supply efficiency of stored red blood cells.Hemoglobin has a special tetramer structure.The binding and dissociation of hemoglobin with oxygen shows a slow and then fast cascade reaction,forming a special S-shaped oxygen binding/dissociation curve.This cascade reaction is called synergistic effect.The index describing the synergistic effect between tetramers is called Hill coefficient（HC）.Bohr effect is also one of the factors affecting the oxygen supply process of red blood cells.Bohr effect refers to the phenomenon that when H⁺increases,the oxygen dissociation curve shifts to the right,the P₅₀ value increases,and the oxygen affinity of hemoglobin decreases.The gradient of H⁺concentration in human body is the basis of Bohr effect.The difference of Bohr effect can be evaluated by sensitivity index（SI）.Synergistic effect and Bohr effect may affect the oxygen supply efficiency of stored red blood cells together with hemoglobin oxygen affinity,and the effects of the above factors should be studied.This study is based on the newly developed hemoglobin detection device BLOODOX-2018 oxygenation dissociation analyzer,and improves the detection of this equipment.Through the detection of many kinds of samples,this paper explores the effects of hemoglobin oxygen affinity,synergistic effect and Bohr effect on oxygen supply efficiency,combined with the upgrade and optimization of the software system,and finally realizes the multi-parameter evaluation of oxygen supply efficiency of stored red blood cells.To understand the changing trend of various parameters and to provide a new understanding for clinical blood transfusion therapy.Chapter 1:oxygen supply efficiency multi-parameter evaluation equipment improvementBLOODOX-2018 oxygenation dissociation analyzer based on visible spectrophotometry and gas control method,simulate the oxygen binding/dissociation process of hemoglobin in vitro,record the oxygen binding/dissociation curve,and obtain the P₅₀ value of the sample to be tested,but it cannot meet the requirements of oxygen supply efficiency multi-parameter evaluation at present.In order to optimize it to meet the requirements of further research,the following studies were carried out:to clarify the impact of sample influencing factors on the test results.Bovine hemoglobin（b Hb）and human stored red blood cells were used as samples to compare the results of1.5mg,3mg,6mg,9mg and 12mg hemoglobin or the corresponding number of red blood cells.The results show that 3mg hemoglobin or the corresponding number of red blood cells can be used as the minimum amount of sample for detection.Human venous blood,canine venous blood and rat venous blood were used as samples to compare the effects of anticoagulants such as heparin sodium,EDTA,3.2%sodium citrate and 3.8%sodium citrate on the test results.The results show that the four anticoagulant components can be used as anticoagulant components detected by oxygenation dissociation analyzer but the anticoagulant components cannot be mixed in the same experiment.The accuracy,stability and testing advantages of the equipment are analyzed.The P₅₀ value of human venous blood was determined simultaneously by oxygenation dissociation analyzer and clinical blood gas analyzer.The results of the two devices are consistent,which proves that the oxygenation dissociation analyzer has the detection accuracy needed for the next work.BHb was repeatedly tested by oxygenation dissociation analyzer for many times,and the average coefficient of variation（CV）between groups was 4.22%,which met the requirement that the CV value of clinical testing equipment was less than 5%.It was confirmed that the oxygenation dissociation analyzer had the detection stability needed for the next experiment.The P₅₀ values of human hemoglobin,bovine blood,b Hb,arterial and venous blood of dogs and rats were simultaneously determined by oxygenation dissociation analyzer and blood gas analyzer.Compared with blood gas analyzer,oxygenation dissociation analyzer has the advantages of wider detection range of samples and not disturbed by other blood gas indexes.The rat model of stored red blood cells was established and the feasibility of oxygenation dissociation analyzer for stored red blood cells was verified.The rat model of stored red blood cells was established,and the hematological indexes,blood oxygen indexes,basic biochemical indexes and hemolysis rate were detected,and the quality of red blood cells was evaluated.The results showed that the metabolic index of stored red blood cells for 14 days was close to that of stored red blood cells for 35 days.The P₅₀values of rat erythrocytes stored for 1 day,3 days and 7 days were measured by oxygenation dissociation analyzer and blood gas analyzer,respectively.The results of blood gas analyzer showed that there was no significant difference in the P₅₀ value of the three time points,while the P₅₀ value of oxygenation dissociation analyzer decreased with the extension of storage time,which was consistent with the literature report.Summary:this chapter determines that the minimum sample volume detected by the oxygenation dissociation analyzer is 3mg hemoglobin or the red blood cells,defines the effects of the four anticoagulants on the detection,defines the accuracy,stability and detection advantages of the equipment,and confirms that it can be used to detect the P₅₀value of stored red blood cells,so as to provide available detection equipment for further research.Chapter 2:the improvement of oxygen supply efficiency multi-parameter evaluation system of oxygen supply efficiencyIn order to study the actual influence of hemoglobin oxygen affinity,synergistic effect and Bohr effect on oxygen supply efficiency,this chapter constructs and calculates the calculation method of oxygen supply efficiency based on different environmental conditions and reference factors by calculating and analyzing the P₅₀value,Hill coefficient and sensitivity index of Han blood,Tibetan blood,b Hb and Dex20-b Hb.The effects of hemoglobin oxygen affinity,synergistic effect and Bohr effect on oxygen supply efficiency were discussed,and a multi-parameter evaluation system of oxygen supply efficiency was established.The specific research contents are as follows:The hemoglobin oxygen affinity(P₅₀ value),synergistic effect（Hill coefficient）and Bohr effect（sensitivity index）were calculated and analyzed for Han blood,Tibetan blood,b Hb and Dex20-b Hb samples.From big to small are:b Hb 29.75 mm Hg±2.37mm Hg,Han blood 24.23 mm Hg±2.56 mm Hg,Tibetan blood 19.77 mm Hg±1.22 mm Hg and Dex20-b Hb 16.35 mm Hg±0.47 mm Hg.The oxygen saturation fitting range of Hill equation was selected by using Han blood samples,and the fitting results of 1%-99%,20%-80%and 40%-60%oxygen saturation intervals were compared.The determination coefficient of the fitting equation obtained from the oxygen saturation fitting range of20%-80%is higher and closer to the results reported in the literature.Then,the above four samples were analyzed in the fitting interval of 20%-80%oxygen saturation,from large to small:b Hb 2.51±0.31,Tibetan blood 2.46±0.07,Han blood 2.27±0.21 and Dex20-b Hb 1.45±0.09,p<0.05.The results showed that the above four samples were analyzed in the fitting interval of 20%-80%oxygen saturation.The occurrence of Bohr effect is simulated by using buffer systems with different p H values,p H=7.2 and p H=7.6 are selected to simulate the acid-base environment of tissue cells and lungs respectively,p H=7.4 simulates the general acid-base environment in vivo,P₅₀values are obtained and sensitivity index is selected to evaluate Bohr effect,and SI=(P_{50（p H=7.2）}-P_{50（p H=7.6）})/P_{50（p H=7.4）}*100%is used to calculate the sensitivity index of the four samples,From big to small:b Hb 41.15%±6.31%,Tibetan blood 35.28%±3.55%,Han blood29.51%±3.19%and Dex20-b Hb 19.35%±2.28%,p<0.05.The method of calculating the oxygen supply efficiency of the samples in different environments is constructed and calculated.First of all,the oxygen partial pressure intervals of simulated plain environment and high-altitude environment are constructed.100mm Hg-40mm Hg and 60mm Hg-30mm Hg correspond to the pulmonary partial pressure of oxygen and tissue cell partial pressure of oxygen respectively,and the oxygen saturation difference between lung and tissue cellsΔSO₂ is used to calculate the efficiency of oxygen supply.Secondly,different calculation systems are constructed,and the oxygen supply efficiency of different samples in different environments is calculated respectively under the influence of single P₅₀ and based on multi-parameter system,and the difference between the two systems is made clear.The results are as follows:Considering the influence of a single P₅₀,the calculated values of oxygen supply efficiency of simulated plain environment are as follows:Han blood 27.00%±1.73%,Tibetan blood 20.24%±2.25%,p<0.01;b Hb 31.91%±0.53%,Dex20-b Hb12.04%±0.43%,p<0.01.The calculated values of oxygen supply efficiency in plateau environment are as follows:24.34%±2.11%for Han blood,22.27%±0.93%for Tibetan blood,p>0.05;b Hb 35.62%±0.44%,Dex20-b Hb 15.95%±0.99%,p<0.01.Based on the analysis of oxygen supply efficiency based on multi-parameter evaluation system,the calculated values of oxygen supply efficiency in simulated plain environment are as follows:29.51%±1.82%of Han blood,27.40%±3.32%of Tibetan blood,p<0.05,48.78%±1.74%of b Hb and 21.40%±1.81%of Dex20-b Hb,p<0.01.The calculated values of oxygen supply efficiency in simulated plateau environment are as follows:26.51%±1.45%for Han blood,30.42%±2.73%for Tibetan blood,p<0.05.For b Hb 53.78%±2.01%,and Dex20-b Hb 30.42%±1.79%,p<0.01.According to the further analysis of the results,considering the influence of single P₅₀,there is no statistical difference in the calculated value of oxygen supply efficiency between Tibetan blood at plain and at high altitude.however,the oxygen supply efficiency analysis based on multi-parameter system shows that the calculated value of oxygen supply efficiency at high altitude is higher than that at plain,which is consistent with the literature report.At the same time,the oxygen supply efficiency analysis system based on multi-parameter evaluation system also covers the effects of hemoglobin oxygen affinity,synergistic effect and Bohr effect on oxygen supply efficiency.Furthermore,the relationships among hemoglobin oxygen affinity,synergistic effect,Bohr effect and oxygen supply efficiency of the four samples were analyzed.The calculated values of oxygen supply efficiency in plain environment and plateau environment were analyzed by multiple regression analysis with P₅₀ value,Hill coefficient and acid-base sensitivity index,respectively.The multiple linear models are as follows:Y=5.501+0.588X₁+10.839X₂+0.032X₃（plain environment）Y’=4.493+0.146X₁+2.152X₂+0.511X₃（plateau environment）Note:X₁,P₅₀;X₂,Hill coefficient,X₃,sensitivity index;Y,ΔSO₂;Y’,ΔSO2’The above results show that hemoglobin oxygen affinity,synergistic effect and Bohr effect affect oxygen supply efficiency together,forming a multi-parameter evaluation system of oxygen supply efficiency.Based on the standardized regression coefficient of the regression equation obtained from the existing data,it is inferred that the synergistic effect has a higher impact on the oxygen supply efficiency of the plain environment,and the Bohr effect has a higher impact on the oxygen supply efficiency of the plateau environment.However,at present,this conclusion is limited to the statistical derivation and analysis of the existing data.In view of the above established method,the software is upgraded and optimized.On the basis of the original data acquisition and control software,the data set and data conversion are formed based on the Visual Basic.NET development language.Through the transduction of the original signal,the fitting module of Hill coefficient,the calculation module of sensitivity index and the calculation module of interval oxygen supply efficiency are constructed to realize the intelligent output of the results.The unified coordinate system and the result database are constructed,and the arbitrary oxygen dissociation curve is transformed into the unified coordinate system according to the same proportion,so that it can intelligently store,call and compare the arbitrary oxygen dissociation curve and data results.Summary:in this chapter,by characterizing the calculation methods of synergistic effect,Bohr effect and oxygen supply efficiency,different types of samples are used to establish a multi-parameter evaluation system of oxygen supply efficiency based on oxygen affinity(P₅₀ value),synergistic effect（Hill coefficient）and Bohr effect（sensitivity index）.Compared with the original single consideration of P₅₀,it can more accurately reflect the change of oxygen supply efficiency.Optimize the software system so that all the results can be obtained and output directly.Chapter 3:analysis of oxygen supply efficiency of rat stored red blood cells based on multi-parameter evaluation systemBased on the established rat red blood cell storage model,the multi-parameter evaluation system is used to analyze its oxygen supply efficiency.The results show that:P₅₀ value:0d 56.42 mm Hg±5.24 mm Hg,1d 49.11 mm Hg±7.02 mm Hg,3d 38.89mm Hg±9.29 mm Hg,7d 28.21 mm Hg±3.27 mm Hg and 14d 25.69 mm Hg±4.88 mm Hg.0d-7d decreased rapidly and 7d-14d tended to be stable,p>0.05,which was basically consistent with that reported in the literature.Hill coefficient:0d 1.98±0.11,1d 2.11±0.06,3d 2.32±0.14,7d 1.99±0.07 and 14d1.88±0.12.0d-3d increased gradually and reached the peak at 3 days,while 3d-14d decreased gradually,p<0.05.Sensitivity index:0d 34.82%±1.17%,1d 46.73%±2.28%,3d 46.73%±2.61%,7d55.82%±3.95%and 14d 24.01%±6.42%.It increased gradually from 0d to 7d,p<0.05,reached the highest at 7d,and decreased at 14d,p<0.05.Calculated value of oxygen supply efficiency:calculated value of oxygen supply efficiency of simulated plain environment（100mm Hg-40mm Hg）:0d 64.69%±4.62%,1d 60.18%±4.71%,3d 53.10%±7.03%,7d 41.18%±1.19%and 14d 30.63%±6.95%.0d-1d was basically unchanged,but 3d-14d decreased,which was only 46.88%of that of 0d at 14 days.The oxygen supply efficiency of red blood cells stored in the simulated plateau environment（60mm Hg-30mm Hg）is lower than that in the plain environment as a whole,and the results of their changes over time are:0d 48.72%±5.88%,1d47.94%±5.78%,3d 45.08%±7.83%,7d 43.77%±7.58%and 14d 35.32%±2.31%,respectively.0d-7d was basically unchanged,7d-14d decreased,p<0.05,and decreased to 70%of 0d at 14d.Summary:stored red blood cells have stable oxygen supply efficiency in the early stage of storage,and begin to decrease in the middle and later stage of storage.the overall trend is different from the change of P₅₀ value.Compared with the plain environment,the oxygen supply efficiency of stored red blood cells in plateau environment decreased as a whole,but the relatively stable time was prolonged.The synergistic effect and Bohr effect of stored erythrocytes were enhanced 3 and 7 days before storage,which was consistent with the change trend of the calculated value of oxygen supply efficiency.Conclusion:based on the BLOODOX-2018 oxygenation dissociation analyzer newly developed by our research group,this study evaluated the changes of rat stored red blood cells under the multi-parameter evaluation system from two aspects:the optimization and evaluation of oxygen supply efficiency multi-parameter evaluation equipment and the establishment of multi-parameter evaluation parameter system of oxygen supply efficiency.First of all,the equipment is improved,and the influencing factors of sample detection and the accuracy,stability and detection advantages of the equipment are studied.Then,through the detection and analysis of Han blood,Tibetan blood,b Hb and Dex20-b Hb,it is clear that hemoglobin oxygen affinity(P₅₀ value)cannot fully reflect the change of oxygen supply efficiency,synergistic effect（Hill coefficient）and Bohr effect（sensitivity index）are both factors affecting oxygen supply efficiency,and a multi-parameter evaluation system of oxygen supply efficiency should be established.The influencing factors were verified by multiple linear regression equation,and the intelligent parameter output software was optimized.Based on the above equipment and multi-parameter evaluation system,the oxygen supply efficiency of stored red blood cells in rats was detected and analyzed.The change trend of oxygen supply efficiency of stored red blood cells in rats was clarified,which provided a new understanding for the further study of oxygen supply efficiency and blood transfusion effect of stored red blood cells.