Affected Factors of Separating Erythrocytes by Discontinuous of Density Gradient Centrifugation

Abstract

To study the affected factors of the separation of red blood cells by discontinuous density gradient method, and to establish the optimal operating conditions of percoll for the separation of erythrocytes. The osmolality of different ratio of Percoll and 1.5 mol/L NaCl were 9:1, 10:1, 11:1, 12:1, 13:1 ratio, was detected respectively. The isotonic osmolality ratio was selected as optimum stock isotonic Percoll solution(SIP). Evaluation criteria: All the different density cells were separated sufficiently by centrifugation, there is little red cell retention and entrainment phenomena. The separation efficacy of different red cell suspension concentration, different centrifugal force, different centrifugal speed, different temperature and different time were compared. The separation efficacy of red cell suspension concentration which not exceed 50% was better than above 50%, efficacy of 4 000×g was superior to 3 000×g and 2 000×g, 2-8 centrifugal acceleration and deceleration centrifugal could obtain the satisfactory efficacy, the 4 °C layered effect was more stable than room temperature, the separation effect of 20 min was better than 15 min and 10 min. Percoll and 1.5 mol/L NaCl 12:1 to 11:1 can be used as stock isotonic percoll solution, preferentially selected 4 °C as centrifugal conditions. When conditions of separation was satisfied, the centrifugal acceleration and deceleration is 2-8, the optimal centrifugal effect can be obtained with the decrease of erythrocyte suspension concentration, with centrifugation time prolonging and centrifugal force increasing.