Zigeuner R E, Riesenberg R, Pohla H, Hofstetter A, Oberneder R
Department of Urology, University of Graz, Graz, Austria.
J Urol. 2000 Nov;164(5):1834-7.
We describe a method to improve tumor cell detection compared to currently available immunocytochemical methods by using immunomagnetic cell enrichment.
Two different methods of immunomagnetic cell enrichment using antibody coated magnetic beads were tested and compared with unenriched immunocytochemistry. One method was positive selection of epithelial cells from mononuclear cells with the antiepithelial antibody BER-EP4 and the other was depletion of mononuclear cells with the antileukocyte antibody CD45. Mononuclear cells were isolated from peripheral blood by density centrifugation and various numbers of tumor cells were added. The 5 different cell lines from urological malignancies used in the study were DU-145, RT-4, CAKI-2, KTCTL-2 and KTCTL-30. Following incubation of cell suspensions with the beads, cell separation was performed in a magnetic field. After centrifugation on glass slides immunocytochemical staining for cytokeratin was performed. A total of 112 experiments were completed and negative controls were obtained.
The number of tumor cells detected by positive selection and depletion was significantly higher than by immunocytochemistry (p <0.001). The median enrichment factor and tumor cell recovery rate for positive selection and depletion were 15.3 and 61.2%, and 13.0 and 57.3%, respectively (not significant). With less than 1 tumor cell suspended in 106 mononuclear cells, the probability of tumor cell detection was 23% for immunocytochemistry alone and 93.3% for both enrichment methods (p <0.01). No false-positive results were observed.
Compared to immunocytochemistry, immunomagnetic cell enrichment significantly improves the sensitivity of detection of epithelial cells added to mononuclear cells. Both methods of enrichment were equally effective and may be important for clinical practice in the future.
我们描述了一种通过免疫磁珠细胞富集来提高肿瘤细胞检测的方法，与目前可用的免疫细胞化学方法相比。
测试了两种使用抗体包被磁珠的免疫磁珠细胞富集方法，并与未富集的免疫细胞化学方法进行比较。一种方法是用抗上皮抗体BER-EP4从单核细胞中阳性选择上皮细胞，另一种方法是用抗白细胞抗体CD45去除单核细胞。通过密度离心从外周血中分离单核细胞，并添加不同数量的肿瘤细胞。本研究中使用的5种泌尿系统恶性肿瘤不同细胞系为DU-145、RT-4、CAKI-2、KTCTL-2和KTCTL-30。细胞悬液与磁珠孵育后，在磁场中进行细胞分离。在载玻片上离心后，进行细胞角蛋白的免疫细胞化学染色。共完成112次实验，并获得阴性对照。
通过阳性选择和去除法检测到的肿瘤细胞数量显著高于免疫细胞化学法（p<0.001）。阳性选择和去除法的中位富集因子和肿瘤细胞回收率分别为15.3和61.2%，以及13.0和57.3%（无显著性差异）。当每106个单核细胞中悬浮的肿瘤细胞少于1个时，单纯免疫细胞化学法检测到肿瘤细胞的概率为23%，而两种富集方法均为93.3%（p<0.01）。未观察到假阳性结果。
与免疫细胞化学相比，免疫磁珠细胞富集显著提高了添加到单核细胞中的上皮细胞的检测灵敏度。两种富集方法同样有效，可能对未来的临床实践具有重要意义。
