Buño Ismael, Nava Paola, Simón Ainhoa, González-Rivera Milagros, Jiménez José Luis, Balsalobre Pascual, Serrano David, Carrión Rafael, Gómez-Pineda Alfonso, Díez-Martín José Luis
BMT Unit, Dep. of Oncology, Hosp. G. U. Gregorio Marañón, Madrid, Spain.
Haematologica. 2005 Oct;90(10):1373-9.
Despite the great utility of chimerism analysis after allogeneic stem cell transplantation, a gold standard method for its quantification has not yet been defined. The objective of the present investigation was to compare the sensitivity (detection limit) and the quantification accuracy of fluorescent in situ hybridization with specific probes for the sex chromosomes (XY-FISH) and multiplex short tandem repeat polymerase chain reaction (STR-PCR) revealed by capillary electrophoresis for the quantification of chimerism after stem cell transplantation.
A first experiment was performed on two sets of artificial cell mixtures from two sex-mismatched healthy donors mixed in different proportions (% male: 100, 75, 50, 25, 10, 5, 3, 1, 0.1, 0). In a second experiment, 58 samples obtained from 10 selected patients with different clinical courses and chimerism evolution after sex-mismatched stem cell transplantation, which had been studied by XY-FISH, were retrospectively analyzed by STR-PCR. In a third experiment, 60 unselected prospective samples belonging to 15 patients (5 of whom had also been included in the retrospective study) were analyzed by both XY-FISH and STR-PCR.
Both techniques showed high quantification accuracy and were highly reproducible. The sensitivity of both approaches reached 1% under standard conditions. Moreover, the use of long injection times for the capillary electrophoresis (30 and 50s vs. the standard 10s) resulted in an increase of sensitivity of the STR-PCR assay up to 0.1%, which has interesting clinical implications.
Considering the high sensitivity and quantification accuracy of multiplex STR-PCR and the fact that this assay is sex-independent and can be applied to virtually all patients, STR-PCR could be considered as the method of choice for chimerism quantification after stem cell transplantation when high sensitivity is not a requirement.
尽管异基因干细胞移植后的嵌合分析具有很大的实用价值，但尚未确定其定量的金标准方法。本研究的目的是比较用于性染色体的特异性探针荧光原位杂交（XY-FISH）和通过毛细管电泳显示的多重短串联重复聚合酶链反应（STR-PCR）在干细胞移植后嵌合定量中的灵敏度（检测限）和定量准确性。
第一个实验是对来自两名性别不匹配的健康供体的两组人工细胞混合物进行不同比例混合（男性比例：100%、75%、50%、25%、10%、5%、3%、1%、0.1%、0%）。在第二个实验中，对10例经过XY-FISH研究的性别不匹配干细胞移植后具有不同临床病程和嵌合演变的患者所获得的58个样本进行STR-PCR回顾性分析。在第三个实验中，对属于15例患者（其中5例也包括在回顾性研究中）的60个未选择的前瞻性样本同时进行XY-FISH和STR-PCR分析。
两种技术均显示出高定量准确性且具有高度可重复性。在标准条件下，两种方法的灵敏度均达到1%。此外，毛细管电泳使用较长进样时间（30秒和50秒，而不是标准的10秒）可使STR-PCR检测的灵敏度提高至0.1%，这具有有趣的临床意义。
考虑到多重STR-PCR的高灵敏度和定量准确性，以及该检测方法与性别无关且几乎可应用于所有患者这一事实，当不需要高灵敏度时，STR-PCR可被视为干细胞移植后嵌合定量的首选方法。
