骨髓活检组织EBER原位杂交检测影响因素分析
|
摘要
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 20-24 h and subsequently wash it with running water for 30 min-1 h. 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.
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 20-24 h and subsequently wash it with running water for 30 min-1 h. 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.
引文
[1]Zhou Y,Attygalle AD,Chuang S,et al.Angioimmunoblastic T-cell lymphoma:histological progression associates with EBV and HHV6B viral load.Br J Haematol,2007,138(1):44-53.
[2]Ha SY,Sung J,Ju H,et al.Epstein-Barr virus-positive nodal peripheral T cell lymphomas:Clinicopathologic and gene expression profilin study.Pathol Res Pract,2013,209(7):448-454.
[3]翁丹枫,张声.改良EDTA脱钙液在骨髓活检组织中染色效果的观察.临床和实验医学杂志,2018,34(4):461-462.
[4]甘启焕,杨清绪.骨髓活检标本脱钙及除酸方法的改良.诊断病理学杂志,2011,18(2):151.
[5]丁伟,王德田,董建强,等.简明病理学技术.杭州:浙江科学技术出版社,2014:186.
[6]吴鸿雁王婷.三种不同脱钙液对乳腺癌骨转移标本染色效果的对比研究.中华病理学杂志,2017,46(8):536-541.
[7]彭大云,张伟.改良EBER原位杂交技术在鼻咽肿瘤病理诊断中的应用.实用医技杂志,2017,24(7):723-724.
[8]Zordan A.Fluorescence in situ hybridization on formalin-fixed,paraffin-embedded tissue sections.Methods Mol Biol,2011,730:189-202.
[2]Ha SY,Sung J,Ju H,et al.Epstein-Barr virus-positive nodal peripheral T cell lymphomas:Clinicopathologic and gene expression profilin study.Pathol Res Pract,2013,209(7):448-454.
[3]翁丹枫,张声.改良EDTA脱钙液在骨髓活检组织中染色效果的观察.临床和实验医学杂志,2018,34(4):461-462.
[4]甘启焕,杨清绪.骨髓活检标本脱钙及除酸方法的改良.诊断病理学杂志,2011,18(2):151.
[5]丁伟,王德田,董建强,等.简明病理学技术.杭州:浙江科学技术出版社,2014:186.
[6]吴鸿雁王婷.三种不同脱钙液对乳腺癌骨转移标本染色效果的对比研究.中华病理学杂志,2017,46(8):536-541.
[7]彭大云,张伟.改良EBER原位杂交技术在鼻咽肿瘤病理诊断中的应用.实用医技杂志,2017,24(7):723-724.
[8]Zordan A.Fluorescence in situ hybridization on formalin-fixed,paraffin-embedded tissue sections.Methods Mol Biol,2011,730:189-202.