The Study Of Correlation Between Red Blood Cell Age And Function

Objectives To study the correlation of cell age and function in the process of the red blood cell normal metabolism. It contributed to provide experimental data for further study of the variation of the function with red blood cells stored in the save process time, calculating the red blood cells function doses of different storage time, achieving quantitative red blood cell transfusion, and improving the therapeutic effect of red blood cell transfusion.Methods 1. The use of discontinuous density gradient separation to divide whole blood into different fractions:we used orthogonal design to analyse separation outcome on different separation conditions, and applied multivariate linear regression analysis to analyse effects on separation of different individual parameters, and optimized separation conditions.2.The use of spectrophotometry and flow cytometry determinated red blood cell age:We divided the whole blood specimen into six fractions with different density(1.085,1.088,1.089,1.091,1.092,1.094g/ml), determinated the red blood cell pyruvate kinase activity of each fraction, and calculated the ratio by pyruvate kinase activity of each fraction with whole blood, then speculated the average cell age of fractions. Meanwhile, we determinated the membrane phosphatidylserine positive expression rate of fractions to reflect the relationship between RBC density and age.3. The determination of red blood cell function:The expression of CR1 on cell membrane in each fraction was determinated by the Flow Cytometry, and the activity of CR1 by erythrocyte rosette test of natural immune adhering tumor cell, and the concentration of 2,3-DPG by spectrophotometry.Results 1. Data indicated that the centrifugal force, centrifugal time, specimen cell concentration, mean corpuscular volume, and individual age influenced the results, in which especially mean corpuscular volume. We chosed the centrifugation conditions was 3500g,20min,4℃. The data also indicated that the mean corpuscular volume of red blood cells with different density reduced from 94.9±3.8fl to 87.2±1.5fl, downed 11.86%. But mean corpuscular hemoglobin concentration increased from 282.5±17.9g/l to 316.0±23.7g/l, increased by 11.86%.2. Data suggested that with the increase in red blood cell density, PS expression rate increased gradually. According to the ratio, red blood cell average age (from low density to high) was 11.5 days,46.0 days,63.8 days, 74.6 days,80.5 days,102.2 days.3. The results of red blood cell related functions test indicated:1) The positive rate of erythrocyte CR1 receptor expression reduced from 73.71±13.62% to 49.13±21.40%; 2) erythrocyte rosette test from 61.0±11.7% to 27.0±8.2%; 3) Among the number of CD35+ in 100 red blood cells(CD35+%), CR1 activity(Er%) and the cell age(D), they had certain relevance in each fraction. The specific relationship can be expressed as the formula Er%= (61.691+0.11×CD35+—0.365×D)/100×100%; 4) The 2,3-diphoglycerate concentration in fractions decreased from 6.15±0.78to3.73±0.48 (mmol/L erythrocytes).Conclusion 1.The discontinuous density gradient separation could divide whole blood red blood cells into several different parts of different cell age, red blood cell density and age were closely related. Combining this method and the technology of use the pyruvate kinase activity to evaluate the average red blood cell age, was contributed to provide methodological foundation for study the correlation of cell age and function.2.View of the preliminary assessment of functional point of erythrocyte CR1 receptor expression, activity changes, and 2,3-DPG concentration were closely related with erythrocyte ages.3. Age and its main function of red blood cells (oxygen-carrying function and immune function) were negatively correlated.