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目的建立神经母细胞瘤(NB)患儿骨髓(BM)微量肿瘤病灶(MRD)的检测方法,分析其结果与预后的关系。方法应用CD45FITC/CD81PE/CD56PECy5单抗组合通过流式细胞术(FCM)检测人NBTGW细胞株的免疫表型,并确定本实验室FCM的敏感度;比较Ⅲ~Ⅳ期NB患儿治疗前及治疗中BM细胞形态学观察与FCM检测BM中的CD45-/CD81+/CD56+细胞阳性率,分析病程中MRD检测与预后的关系。结果①TGW细胞株表达CD56和CD81,不表达CD45,本实验室FCM敏感度为1×10-4。②58例患儿共144份标本中BM细胞形态学观察分析提示BM转移者23份,FCM检测显示表达CD45-/CD81+/CD56+细胞的标本数62份,BM细胞形态学观察和FCM法阳性检出率差异有统计学意义(P<0·01)。③比较平均化疗4个疗程后BMMRD结果与预后关系:31例患儿初诊时BMMRD检测阳性,化疗4个疗程后11例患儿BMMRD转阴,随访至今无复发及进展,无瘤生存率(DFS)中位时间23个月;另20例患儿化疗4个疗程后BMMRD仍为阳性,其中11例患儿复发或进展,包括1例死亡,两组差异有统计学意义(P<0·01)。④化疗后移植患儿外周血干细胞(PBSC)采集前BMMRD结果与预后的相关性:共有19例患儿接受化疗后行PBSC移植,14例患儿采集PBSC时BMMRD为阴性,其中2例患儿复发,自移植后始DFS中位时间9个月;5例患儿采集PBSC时BMMRD为阳性,均复发,两组差异有统计学意义(P<0·05)。结论运用FCM检测NB患儿BMMRD敏感度高、特异性强,可协助NB患儿的诊断和评估临床疗效,BMMRD残留与NB预后相关。
Objective To establish a method for detection of micro-tumorous lesions (MRD) in bone marrow (BM) of children with neuroblastoma (NB), and to analyze the relationship between outcome and prognosis. Methods The immunophenotypes of human NBTGW cell lines were detected by flow cytometry (FCM) using the combination of CD45FITC / CD81PE / CD56PECy5 monoclonal antibody and the sensitivity of FCM in our laboratory was determined. Before treatment and treatment In the observation of BM cell morphology and the positive rate of CD45- / CD81 + / CD56 + cells detected by FCM in BM, the relationship between MRD detection and prognosis in the course of disease was analyzed. Results ① TGW cells expressed CD56 and CD81, but did not express CD45. The sensitivity of FCM in this laboratory was 1 × 10-4. ② The morphological observation of BM cells in 144 samples of 58 children showed that there were 23 BM metastases in all, and FCM showed that there were 62 samples expressing CD45- / CD81 + / CD56 + cells. The morphological observation of BM cells and the positive results of FCM The difference was statistically significant (P <0.01). The results of BMMRD showed that the BMMRD was positive after 4 cycles of chemotherapy. The BMMRD of 11 cases was negative after 4 cycles of chemotherapy. There was no recurrence and progression to follow-up, and the tumor-free survival rate (DFS) ) For a median of 23 months. In the other 20 cases, BMMRD was still positive after 4 courses of chemotherapy, of which 11 cases had recurrence or progression, including 1 case of death, the difference was statistically significant (P <0.01) ). (4) The correlation between the results of BMMRD and prognosis before the transplantation of peripheral blood stem cells (PBSC) in children after chemotherapy: A total of 19 children underwent PBSC after chemotherapy, and the BMMRD was negative in 14 children with PBSC, of which 2 children The recurrence and median DFS were 9 months after transplantation. The BMMRD was positive in 5 children with PBSC recurrence and the difference was statistically significant (P <0.05). Conclusion The detection of BMMRD using FCM in children with NB is sensitive and specific. It can be used to diagnose and evaluate the clinical efficacy of NB in children. The residual of BMMRD is correlated with the prognosis of NB.