Silvestri F, Banavali S, Savignano C, Preisler H D, Baccarani M
Department of Medical and Morphological Researches, Udine University School of Medicine, Italy.
Int J Artif Organs. 1993 Dec;16 Suppl 5:96-101.
The aim of the present study was to develop immunoadsorption techniques for purifying CD34+ cells from the peripheral blood (PB) and the bone marrow (BM). First, we compared two methods for isolating CD34+ cells from patients with acute non-lymphocytic leukemia. Twenty-two samples (17 PB, 5 BM) were obtained from 19 patients, were density cut and, after incubation with My10 antibody, were separated by panning or by immunomagnetic beads. Beads allowed a significantly better separation, either in terms of purification of CD34+ cells (85.5% +/- 11.1% vs 55.7 +/- 23.8%, p = 0.003) or in terms of depletion of CD34+ cells from the negative fractions (3.9 +/- 7.6 vs 30.9 +/- 25.1%, p = 0.008). In a second study, 2 BMs from healthy subjects and 1 BM and 2 PBs from chronic myeloid leukemia patients were separated using immunomagnetic beads. The results confirmed the previous study the overall frequency of CD34+ cells in the isolated positive fractions was 85.0 +/- 10.6% (with a recovery of 64.0 +/- 5.7%), while it was only 2.7 +/- 6.6% in the negative fractions. In particular the purity in the two normal BMs was respectively 86 and 97%. According to these results, the great majority of clonogenic cells was separated in the CD34+ fractions. Chymopapain, that was used to detach the beads from the cells, was shown to be non-toxic to the clonogenic cells. Positive selection of CD34+ cells with immunomagnetic beads and chymopapain is a useful method for isolating almost pure progenitors from the PB and the BM for experimental use and is under investigation for clinical applications.
本研究的目的是开发用于从外周血（PB）和骨髓（BM）中纯化CD34+细胞的免疫吸附技术。首先，我们比较了从急性非淋巴细胞白血病患者中分离CD34+细胞的两种方法。从19例患者中获取了22份样本（17份PB，5份BM），进行密度梯度离心，与My10抗体孵育后，通过淘选法或免疫磁珠法进行分离。无论是从CD34+细胞的纯化方面（85.5%±11.1%对55.7±23.8%，p = 0.003），还是从阴性组分中CD34+细胞的去除方面（3.9±7.6对30.9±25.1%，p = 0.008）来看，磁珠都能实现显著更好的分离效果。在第二项研究中，使用免疫磁珠对来自健康受试者的2份BM以及来自慢性髓性白血病患者的1份BM和2份PB进行了分离。结果证实了先前的研究，即分离出的阳性组分中CD34+细胞的总体频率为85.0±10.6%（回收率为64.0±5.7%），而阴性组分中仅为2.7±6.6%。特别是两份正常BM中的纯度分别为86%和97%。根据这些结果，绝大多数克隆形成细胞在CD34+组分中被分离出来。用于从细胞上分离磁珠的木瓜凝乳蛋白酶对克隆形成细胞无毒。用免疫磁珠和木瓜凝乳蛋白酶对CD34+细胞进行阳性选择是一种从PB和BM中分离出几乎纯的祖细胞用于实验的有用方法，目前正在进行临床应用研究。
