使用甲基噻唑四氮唑法检测细胞耐药性的急性淋巴细胞白血病样本中污染的单核细胞。

Mononuclear cells contaminating acute lymphoblastic leukaemic samples tested for cellular drug resistance using the methyl-thiazol-tetrazolium assay.

作者信息

Kaspers G J, Veerman A J, Pieters R, Broekema G J, Huismans D R, Kazemier K M, Loonen A H, Rottier M A, van Zantwijk C H, Hählen K

机构信息

Department of Paediatrics, Free University Hospital, Amsterdam, The Netherlands.

出版信息

Br J Cancer. 1994 Dec;70(6):1047-52. doi: 10.1038/bjc.1994.446.

DOI:10.1038/bjc.1994.446

PMID:7981053

原文链接:https://pmc.ncbi.nlm.nih.gov/articles/PMC2033662/

Abstract

The methyl-thiazol-tetrazolium (MTT) assay is a drug resistance assay which cannot discriminate between malignant and non-malignant cells. We previously reported that samples with > or = 80% leukaemic cells at the start of culture give similar results in the MTT assay and the differential staining cytotoxicity assay, in which a discrimination between malignant and non-malignant cells can be made. However, the percentage of leukaemic cells may change during culture, which might affect the results of the MTT assay. We studied 106 untreated childhood acute lymphoblastic leukemia (ALL) samples with > or = 80% leukaemic cells at the start of culture. This percentage decreased below 80% in 28%, and below 70% in 13%, of the samples after 4 days of culture. A decrease below 70% occurred more often in case of 80-89% leukaemic cells (9/29) than in case of > or = 90% leukaemic cells at the start of culture (5/77, P = 0.0009). Samples with < 70% leukaemic cells after culture were significantly more resistant to 6 out of 13 drugs, and showed a trend towards being more resistant to two more drugs, than samples with > or = 80% leukaemic cells. No such differences were seen between samples with 70-79% and samples with > or = 80% leukaemic cells after culture. We next studied in another 30 ALL samples whether contaminating mononuclear cells could be removed by using immunoamagnetic beads. Using a beads to target cell ratio of 10:1, the percentage of leukaemic cells increased from mean 72% (s.d. 9.3%) to mean 87% (s.d. 6.7%), with an absolute increase of 2-35%. The recovery of leukaemic cells was mean 82.1% (range 56-100%, s.d. 14.0%). The procedure itself did not influence the results of the MTT assay in three samples containing only leukaemic cells. We conclude that it is important to determine the percentage of leukaemic cells at the start and at the end of the MTT assay and similar drug resistance assays. Contaminating mononuclear cells can be successfully removed from ALL samples using immunomagnetic beads. This approach may increase the number of leukaemic samples which can be evaluated for cellular drug resistance with the MTT assay or a similar cell culture drug resistance assay.

摘要

甲基噻唑四氮唑（MTT）法是一种耐药性检测方法，它无法区分恶性细胞和非恶性细胞。我们之前报道过，培养开始时白血病细胞比例≥80%的样本，在MTT法和鉴别染色细胞毒性试验中结果相似，后者可以区分恶性细胞和非恶性细胞。然而，白血病细胞的比例在培养过程中可能会发生变化，这可能会影响MTT法的结果。我们研究了106例培养开始时白血病细胞比例≥80%的未经治疗的儿童急性淋巴细胞白血病（ALL）样本。培养4天后，28%的样本中该比例降至80%以下，13%的样本降至70%以下。培养开始时白血病细胞比例为80 - 89%的样本（9/29）比白血病细胞比例≥90%的样本（5/77，P = 0.0009）更常出现降至70%以下的情况。培养后白血病细胞比例<70%的样本对13种药物中的6种药物的耐药性明显更高，并且对另外两种药物也有耐药性增加的趋势，相比之下，白血病细胞比例≥80%的样本则不然。培养后白血病细胞比例为70 - 79%的样本与白血病细胞比例≥80%的样本之间未观察到此类差异。接下来，我们在另外30例ALL样本中研究了是否可以使用免疫磁珠去除污染的单核细胞。使用磁珠与靶细胞比例为10:1时，白血病细胞比例从平均72%（标准差9.3%）增加到平均87%（标准差6.7%），绝对增加2 - 35%。白血病细胞的回收率平均为82.1%（范围56 - 100%，标准差14.0%）。该操作本身并未影响三个仅含白血病细胞的样本的MTT法结果。我们得出结论，在MTT法及类似的耐药性检测开始时和结束时确定白血病细胞的比例很重要。使用免疫磁珠可以成功地从ALL样本中去除污染的单核细胞。这种方法可能会增加可以用MTT法或类似的细胞培养耐药性检测评估细胞耐药性的白血病样本数量。