Humblet Y, Lefebvre P, Jacques J L, Bosly A, Feyens A M, Sekhavat M, Agaliotis D, Symann M
Ludwig Institute for Cancer Research, Brussels, Belgium.
Bone Marrow Transplant. 1988 Jan;3(1):63-7.
To perform an optimal ex vivo bone marrow purge, it is necessary to concentrate the bone marrow progenitor cells and to eliminate both the red blood cells and the polymorphonuclear leucocytes. To achieve this goal which cannot be accomplished by using the Haemonetics model 30 alone, we used the Haemonetics model 30 and a density gradient together in a two-step procedure. In the first step we obtained the buffy coat from original bone marrow grafts and in the second we reintroduced these buffy coats into the Haemonetics bowl followed by Percoll, adjusted to 1.079 g/ml, at 5 ml/min in order to recover the light density mononuclear cells. After this second step, the mean volume of the marrow and the RBC and nucleated cell contaminations were reduced to 9%, 0.96% and 16% of their original values the unseparated bone marrow, respectively. This was far better than the values obtained after the first step (volume: 19%; RBC: 10%; nucleated cells: 54%). The CFU-GM recoveries after the first and second steps were 71% and 70% of the original samples, respectively. The entire procedure lasted between 75 and 150 min. At this time, 17 of the 24 patients whose bone marrow was separated using Percoll gradient in the Haemonetics bowl have been grafted. Thirteen of these 17 patients had an evaluable haematological recovery which was complete and rapid for all but one patient with acute myeloid leukaemia. These results demonstrate that the introduction of a density gradient into the Haemonetics model 30 bowl is possible and effective. The reduction in total volume and cell number permits ex vivo purging, without decreasing the grafting capability.
为了进行最佳的体外骨髓净化，有必要浓缩骨髓祖细胞并清除红细胞和多形核白细胞。为实现这一仅使用Haemonetics 30型仪器无法完成的目标，我们采用两步法，将Haemonetics 30型仪器与密度梯度结合使用。第一步，我们从原始骨髓移植物中获取血沉棕黄层，第二步，我们将这些血沉棕黄层重新引入Haemonetics仪器的碗状容器中，随后以5毫升/分钟的速度加入调整至1.079克/毫升的 Percoll，以回收低密度单核细胞。经过第二步后，骨髓的平均体积以及红细胞和有核细胞的污染分别降至未分离骨髓原始值的9%、0.96%和16%。这远比第一步后获得的值要好（体积：19%；红细胞：10%；有核细胞：54%）。第一步和第二步后的CFU-GM回收率分别为原始样本的71%和70%。整个过程持续75至150分钟。此时，在24例使用Haemonetics仪器碗状容器中的Percoll梯度分离骨髓的患者中，已有17例进行了移植。这17例患者中有13例具有可评估的血液学恢复情况，除1例急性髓系白血病患者外，其他患者的恢复均完整且迅速。这些结果表明，在Haemonetics 30型仪器的碗状容器中引入密度梯度是可行且有效的。总体积和细胞数量的减少允许进行体外净化，而不会降低移植能力。
