Hirn-Scavennec J, Brailly H, Maraninchi D, Mannoni P, Mawas C, Delaage M
Immunotech, Marseille, France.
Transplantation. 1988 Oct;46(4):558-63. doi: 10.1097/00007890-198810000-00018.
A new in vitro immunophysical method of removing leukemia or lymphoma cells from autologous bone marrow is described. This new technique makes use of low-density polypropylene beads (density: 0.91) coated with a monoclonal antibody anti-CALLA (antibody ALB2). To ascertain its ability to selectively remove human B/pre-B hematopoietic cells, this technique was applied to normal human bone marrow cell suspensions contaminated with 1-5% of tumor cells. Samples were incubated with the floating beads at 4 degrees C on a rotating wheel for 60 min, followed by a 10-min decantation period, after which the beads bearing the tumor cells floated on the surface, whereas unbound normal marrow cells remained in suspension and were easily recovered free of beads. To demonstrate the feasibility of our method, 2 types of assays were carried out, one using target cell radiolabeled with 111indium, and the other a clonogenic assay. The first assays were to calibrate the different parameters (cellular density, quantity of beads, incubation time) with tumor cell lines: Namalwa (CALLA+) and Molt 4 (CALLA-). These 2 cells lines being able to clone, it is hard to envisage clonogenic assays. In this case, it is very hazardous to evaluate correctly the remaining clonogenic units of Namalwa cells. It is why radiolabelling assays were used for these first experiments. The second assays were to study a model close to the clinical setting and to control the safety of the beads on normal bone marrow cells. In this case, the mixture experiments in which only Namalwa cells were able to clone were evaluated with clonogenic assays, which are more sensitive than radiolabeling assays. A 3- to 4-log reduction of tumor load was achieved with 1-step treatment, and an average of 5-log depletion was obtained by repeating the process twice, as ascertained by the clonogenic assay. Viability, average recovery of nucleated cells, and stem cells potential following the purge were excellent.
本文描述了一种从自体骨髓中去除白血病或淋巴瘤细胞的新型体外免疫物理方法。这项新技术利用了包被有抗CALLA单克隆抗体（抗体ALB2）的低密度聚丙烯微珠（密度：0.91）。为确定其选择性去除人B/前B造血细胞的能力，该技术被应用于被1%-5%肿瘤细胞污染的正常人骨髓细胞悬液。样本在4℃下于转轮上与漂浮微珠孵育60分钟，随后倾析10分钟，之后携带肿瘤细胞的微珠漂浮在表面，而未结合的正常骨髓细胞仍悬浮，且很容易无微珠回收。为证明我们方法的可行性，进行了两种类型的检测，一种使用用铟-111放射性标记的靶细胞，另一种是克隆形成检测。首次检测是用肿瘤细胞系Namalwa（CALLA阳性）和Molt 4（CALLA阴性）校准不同参数（细胞密度、微珠数量、孵育时间）。这两种细胞系都能够克隆，因此难以设想克隆形成检测。在这种情况下，正确评估Namalwa细胞剩余的克隆形成单位非常危险。这就是为什么在这些首次实验中使用放射性标记检测的原因。第二次检测是研究一个接近临床情况的模型，并控制微珠对正常骨髓细胞的安全性。在这种情况下，仅Namalwa细胞能够克隆的混合实验用克隆形成检测进行评估，该检测比放射性标记检测更敏感。通过一步处理实现了肿瘤负荷3至4个对数级的降低，通过重复该过程两次平均获得了5个对数级的清除，这通过克隆形成检测得以确定。清除后细胞活力、有核细胞的平均回收率和干细胞潜力都非常好。
