流式细胞技术与PCR技术联用筛选儿童急性淋巴细胞白血病微量残留病标记

Tandem application of flow cytometry and polymerase chain reaction for choice targets of minimal residual disease in childhood acute lymphoblastic leukemia

摘要:

目的：微量残留病（MRD）监测是儿童急性淋巴细胞白血病（ALL）早期治疗反应中最重要的预后因素之一。目前常用的MRD检测的方法主要有流式细胞术和PCR技术，但单用一种方法均不能为所有的患儿找到合适的监测标记，该研究探讨两种方法联合应用是否能为大多数ALL患儿找到合适的MRD监测标记。方法：联合应用流式细胞术和PCR技术筛选上海儿童医学中心2001年9月至2003年10月126例新发ALL患儿骨髓标本的异常免疫表型及抗原受体基因重排。结果：①106例B系-ALL患儿骨髓标本用四色流式细胞术进行了MRD免疫表型标记的筛选，其中11例标本未筛选出监测标记，阳性率为89.6％；有1个监测标记的标本为11例（11.6％），至少有两个标记的标本占88.4％。②PCR技术筛选27例ALL骨髓标本抗原受体基因重排，26例至少有一个标记（占96.3％），其中9例（34.6％）骨髓标本只有一个监测标记，17例（65.4%）至少有两个监测标记。在T系-ALL骨髓标本中，以TCRVγⅠ-Jγ1.3/2.3阳性最多，双等位基因重排发生阳性率较高；系性交叉抗原表达在B系-ALL骨髓标本中表达较高，达57.1％（4/7）。③两种方法联用能为121例（96.0％）的患儿提供合适的筛选指标。结论：流式细胞技术检测异常免疫表型与PCR技术检测抗原受体基因重排联用，可为绝大多数的ALL患儿找到合适的MRD监测指标；在抗原受体基因重排中，存在系性交叉抗原表达及双等位基因重排。［中国当代儿科杂志，2009，11（4）：246-250］

Abstract:

OBJECTIVE: Minimal residual disease (MRD) is one of the most important prognostic factors in childhood acute lymphoblastic leukemia (ALL). Flow cytometry and PCR are two common techniques for examining MRD in ALL. This study aimed to identify MRD targets by tandem application of both techniques in children with ALL. METHODS: From September 2001 to October 2003, 126 children with newly diagnosed ALL were enrolled on the treatment protocol ALL-XH-99. Tandem application of flow cytometry and PCR was performed to identify MRD targets in these patients. RESULTS:① Using sets of combined antibodies, immunophenotypic expression of leukemia cells was observed in 95 of 106 B-lineage ALL cases (89.6%). Only one aberrant immunophenotype was observed in 11 cases (11.6%) and most patients with B-lineage ALL (88.4%) expressed at least two suitable targets. ② Using PCR technique, T-cell receptor (TCR) or immunoglobulin gene rearrangements were identified in 26 of 27 patients (96.3%). Two or more monoclonal/ bi-allelic gene rearrangements were identified in 17 cases (65.4%). The majority (70%) of T-lineage ALL cases contained TCRVγⅠ-Jγ1.3/2.3. Cross-lineage TCR rearrangements were found in 57.1% of cases with B-lineage ALL. ③ Suitable MRD targets of immunophenotypic abnormalities or antigen receptor gene rearrangements were detected in 121 patients (96.0%). CONCLUSIONS: MRD targets were identified using tandem application of flow cytometry and PCR in almost of children with ALL. Cross-lineage TCR rearrangements and bi-allelic gene rearrangements were observed in many patients.［Chin J Contemp Pediatr, 2009, 11 (4):246-250］