骨髓活检组织EBER原位杂交检测影响因素分析

Analysis for the influencing factors of bone marrow biopsy detection via EBER in situ hybridization

目的探讨骨髓活检组织行EBV encoded RNA(EBER)原位杂交检测的影响因素,优化检测条件以期提高骨髓活检组织中EB 病毒的检出率。方法收集35 例EB 病毒相关疾病的骨髓活检标本,通过对比实验,比较不同脱钙方法、不同蛋白酶K 消化条件、不同抗体孵育温度下的骨髓活检组织行EBER 原位杂交检测的切片质量、脱片率及染色质量情况。结果脱钙以运用改良EDTA 脱钙液脱钙20~24h 后流水冲洗30min^1h 最佳;蛋白酶K 消化时间为9min 的组织切片脱片率低,杂交效果最好,阳性细胞着色深,定位准确;在37℃条件下进行抗体孵育杂交染色质量为佳。结论选取合适的脱钙方式,延长蛋白酶K 消化时间,选择最佳抗体孵育温度,可降低脱片率,显著提高骨髓活检组织行EBER 原位杂交检测的染色质量,为EBV 相关疾病的诊断和鉴别诊断提供重要依据。

Objective To investigate the influencing factors of EBV encoded RNA (EBER) in situ hybridization detection in bone marrow biopsy, and to optimize the detection conditions so as to improve the detection rate of EB virus in bone marrow biopsy. Methods Bone marrow biopsy specimens of 35 cases with EBV-related diseases were collected, and the quality of section, detachment rate and dyeing quality of bone marrow biopsy tissues under different conditions, namely different decalcification methods, different protease K digestion conditions, and different antibody incubation temperatures, were compared through comparative experiments. Results The optimal decalcification method was to decalcify the biopsy in modified EDTA decalcification solution for 20-24h and subsequently wash it with running water for 30min-1h. The detachment rate of tissue sections with 9-minute digestion by protease K was low, its hybridization effect was the best, the positive cells were deeply stained and the location was accurate. The hybrid dyeing quality is better under the condition of antibody incubation at 37℃. Conclusion By selecting the appropriate decalcification method, appropriately prolonging the digestion time of protease K and selecting the optimal antibody incubation temperature, the rate of decalcification can be reduced, and the dyeing quality of bone marrow biopsy tissues by EBER in situ hybridization can be significantly improved, which provides an important basis for the diagnosis and differential diagnosis of EBV-related diseases.