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首页> 外文期刊>Bone marrow transplantation >Selective loss of progenitor subsets following clinical CD34+ cell enrichment by magnetic field, magnetic beads or chromatography separation.
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Selective loss of progenitor subsets following clinical CD34+ cell enrichment by magnetic field, magnetic beads or chromatography separation.

机译：通过磁场，磁珠或色谱分离富集临床CD34 +细胞后祖细胞的选择性损失。

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In this preclinical evaluation we have compared the efficacy of three clinical CD34+enrichment procedures with respect to purity, yield and recovery, as well as risk of selective loss of CD34+ lineage-specific subsets. The three devices work by different principles and have several different manipulation steps: The magnetic field separator uses paramagnetic iron-dextran particles; the magnetic microbead selection is based on the advantage of a large surface area for immobilisation of the monoclonal antibody within a very small volume; the original immunoabsorption technique is based on the use of biotinylated antibody applied to a column of avidin-coated sephadex beads. The results of this evaluation gave a median purity 96% (88-98%), 86% (62-97%), and 49% (18-85%), and median yield of 65% (54-100%), 40% (21-74%), and 30% (8-55%), respectively. Subset analysis recognised a selective loss of CD34+/61+ after enrichment, most likely due to class I-II antibodies used for the enrichment step or, alternatively, nonspecific binding of megakaryocytic progenitors. Tumour cell spiking experiments on a clinical scale documented an expected 2-4 log reduction resulting in a number of potentially malignant cells in the CD34 enriched product. Our data support four major conclusions: First, that magnetic field separation is superior to magnetic beads and chromatography selection, mainly due to the risk of cell loss and insufficient recovery with the two latter methods. Second, that late differentiated progenitors with CD34 class III epitopes present are lost during the enrichment procedures. The third major conclusion is that chromatography selection results in a selective loss of CD34bright cells, which are most likely uncommitted early progenitors. This was an unexpected finding which may be a consequence of an imbalance between the strong forces between biotin-avidin and insufficient physical manipulation for CD34+ cell release. Finally, the data document that CD34 selection alone is an inappropriate way to eliminate tumour cells due to the uncontrolled variables and the inconsistent outcome. The only products which can be expected to be purged free of tumour cells are the ones with very minimal (<10-5) contamination in the starting products, ie products documented tumour free with the most sensitive techniques for quantitation. If this is not the case, the optimal purging strategy may be a two-step procedure including CD34 selection and subsequent depletion of the tumour cells in question.
机译：在这项临床前评估中，我们比较了三种临床CD34 +富集程序在纯度，产量和回收率以及选择性丧失CD34 +谱系特异性亚群的风险方面的功效。这三种设备采用不同的原理工作，并具有几个不同的操作步骤：磁场分离器使用顺磁性葡聚糖颗粒;磁性微珠的选择是基于将单克隆抗体固定在很小体积内的较大表面积的优势。最初的免疫吸收技术是基于将生物素化抗体应用于亲和素包被的葡聚糖微珠柱上。评估结果表明，纯度中位数为96％（88-98％），86％（62-97％）和49％（18-85％），产率中位数为65％（54-100％）， 40％（21-74％）和30％（8-55％）。子集分析确认了富集后CD34 + / 61 +的选择性丢失，这很可能是由于富集步骤中使用的I-II类抗体或巨核细胞祖细胞的非特异性结合所致。临床规模的肿瘤细胞加标实验记录了预期的2-4 log减少，从而在富含CD34的产品中导致了许多潜在的恶性细胞。我们的数据支持四个主要结论：首先，磁场分离优于磁珠和色谱选择，这主要是由于后两种方法具有细胞损失和回收不足的风险。其次，在富集过程中丢失了具有CD34 III类表位的晚期分化祖细胞。第三个主要结论是，色谱选择导致CD34明亮细胞的选择性丢失，而CD34明亮细胞很可能是未定型的早期祖细胞。这是一个出乎意料的发现，可能是由于生物素-亲和素之间的强大力量与CD34 +细胞释放的物理操作不充分之间的不平衡所致。最后，数据文件指出，由于不受控制的变量和不一致的结果，仅CD34的选择是消除肿瘤细胞的不合适方法。可以预期清除出的没有肿瘤细胞的唯一产品是起始产品中污染极小（<10-5）的产品，即使用最敏感的定量技术证明没有肿瘤的产品。如果不是这种情况，则最佳清除策略可能是两步操作，包括选择CD34和随后清除相关肿瘤细胞。

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