甲酸表面脱钙法在骨髓活检荧光原位杂交制片中的应用_滕孝静.pdf

临床和实验医学杂志，2021，20(17):1866 1869 13 刘盛，张维波，向斌，等 GATA 6，Dkk 1 及 bFGF 在垂体腺瘤中的表达及与预后的相关性J 临床误诊误治，2022，35(1):4347 14 陈辉，茅建娅，马震，等 Gal 1、GPX3 及 Tg 水平与甲状腺癌病情严重程度的关系分析J 实用癌症杂志，2022，37(8):1276 1279 15 刘亚琪，郑承红 mi 205 通过靶向调控 Wnt 5a 抑制乳头状甲状腺癌的细胞增殖J 基础医学与临床，2021，41(3):358 363(收稿日期:2022 11 28)DOI:10 3969/j issn 1671 4695 2023 07 024文章编号:1671 4695(2023)07 0762 05甲酸表面脱钙法在骨髓活检荧光原位杂交制片中的应用滕孝静刘伟王凤李颖鸿毕阔孙岚(首都医科大学附属北京友谊医院病理科北京100050)【摘要】目的对骨髓活检组织使用甲酸溶液进行表面脱钙，提高其石蜡切片荧光原位杂交检测的成功率，为骨髓内淋巴瘤的精确诊治及预后判断提供分子生物学依据。方法回顾性收集 2015 年 1 月至 2022 年 5 月间首都医科大学附属北京友谊医院病理科骨髓活检组织 58 例，其中套细胞淋巴瘤 33 例，弥漫大 B 细胞淋巴瘤 21 例，滤泡性淋巴瘤 4例。根据常规制片前所用脱钙液和脱钙方式的不同，将实验分为 3 组:硝酸传统脱钙组(DG1)、硝酸表面脱钙组(DG2)和甲酸表面脱钙组(DG3)。其中 DG2 组和 DG3 组蜡块在荧光原位杂交制片前，需放入 50%甲酸溶液中二次脱钙 10min，流水冲洗 10 min。之后对 3 组蜡块进行连续切片，每例捞取组织面 2 张，分别用于苏木素和伊红染色和杂交检测。结果DG1 组、DG2 组和 DG3 组杂交的成功率分别为 5 6%(1/18)、94 7%(18/19)和 95 2%(20/21)。与 DG1 组相比，DG2 组和 DG3 组杂交成功率明显升高，差异有统计学意义(P 0 05)。DG2 组和 DG3 组成功率之间差异无统计学意义(P 0 05)。DG2 组 13 例 MCL 和 1 例 FL、DG3 组 8 例 MCL 和 2 例 FL 中的 CCND1/IGH 和 BCL2/IGH 融合探针检测均为阳性。DG1 组1 例 DLBCL、DG2 组5 例 DLBCL、DG3 组11 例 DLBCL 细胞内 MYC 基因均未出现异常断裂信号，表现为两个红绿融合的黄色信号。杂交成功的切片上组织结构完整，细胞轮廓清楚，信号定位准确。结论甲酸表面脱钙法脱钙速度快，荧光原位杂交检测成功率高，结果准确。甲酸代替硝酸，配制安全而且容易购买。该法在骨髓活检FISH 制片中的应用具有重要的临床价值，应用前景广阔，值得推广。【关键词】原位杂交荧光骨髓活检淋巴瘤Application of formic acid surface decalcification in fluorescence in situ hybridization section preparation of bone marrow biopsy TENG Xi-ao jing，LIU Wei，WANG Feng，et al Department of Pathology，Beijing Friendship Hospital，Capital Medical University，Beijing 100050，China【Abstract】ObjectiveA method of formic acid surface decalcification in bone marrow biopsy specimen was established to improve thesuccess rate of fluorescence in situ hybridization detection of paraffin sections，and provide molecular biological basis for accurate diagnosis，treat-ment and prognosis for bone marrow lymphoma MethodsFrom January 2015 to May 2022，fifty eight cases of bone marrow biopsy tissues fromthe Department of Pathology，Beijing Friendship Hospital，Capital Medical University were retrospectively collected，including 33 mantle cell lym-phoma，21 diffuse large B cell lymphoma and 4 follicular lymphoma According to the difference of decalcification solution and decalcificationmethod before routine preparation，the experiment was divided into three groups:nitric acid traditional decalcification group(DG1)，nitric acidsurface decalcification group(DG2)and formic acid surface decalcification group(DG3)Before fluorescence in situ hybridization，blocks inDG2 and DG3 groups were placed in 50%formic acid solution for a second decalcification for 10 min and washed under water for 10 min Afterthat，the blocks from three groups were sliced continuously，and two tissue slices were extracted from each case for HE staining and hybridizationdetection respectively esultsThe success rates of hybridization in groups DG1，DG2 and DG3 were 5 6%(1/18)，94 7%(18/19)and95 2%(20/21)，respectively Compared with DG1 group，the success rate of hybridization in DG2 and DG3 groups was significantly increased，and the difference was statistically significant(P 0 05)There was no statistically significant difference of the success rate between DG2 groupand DG3 group(P 005)Fusion probe detection of CCND1/IGH and BCL2/IGH in 13 MCL and 1 FL in DG2 group and 8 MCL and 2 FL inDG3 group were positive The MYC gene of 1 DLBCL in DG1 group，5 DLBCL in DG2 group，and 11 DLBCL in DG3 group showed no abnormalbreak signal，which showed two yellow signals In the successful sections，the tissue structure was complete，the cell outline was clear，and thesignal location was accurate ConclusionThe method of formic acid surface decalcification in bone marrow biopsy has the advantage of fast decal-cification speed，high success rate and accurate results of fluorescence in situ hybridization Formic acid replaces nitric acid，which is safe andeasy to purchase So the application of this method has important clinical value in accurate diagnosis，treatment and prognosis of bone marrow lym-phoma，and it is worth promoting【Key words】In situ hybridization;Fluorescence;Bone marrow biopsies;Lymphoma基金项目:国家重点研发计划“老年人多病共患临床大数据与生物样本库综合管理共享平台建设”项目资助(编号:2020YFC2004800)淋巴瘤是起源于淋巴结和结外淋巴组织的一组恶性肿瘤，常以淋巴结肿大或局部肿块先发，后可累及骨髓。骨髓活检(bone marrow biopsy，BMB)是诊断骨髓内淋巴瘤的主要组织形式之一，一小段组织中除含有娇嫩的淋巴造血细胞、软组织，还含有坚硬的骨质，必须将骨质脱钙后才能制成完整的石蜡切片进行病理检测。酸性脱钙液(硝酸、盐酸、甲酸等)因脱钙速度快，能够满足267Journal of Clinical and Experimental Medicine Vol22，No7Apr2023临床快速发出形态学初诊报告而在病理科广泛应用1 2。荧光原位杂交(fluorescence in situ hybridiza-tion，FISH)技术是一项能将组织、细胞的形态学改变与染色体/基因异常有机结合的一项分子细胞遗传学技术，在淋巴瘤、肺癌、胃癌、乳腺癌等肿瘤的诊治及预后判断中具有重要的指导意义3，可用于大多数组织石蜡切片中。但在酸性溶液脱钙的 BMB 石蜡切片上，FISH不易取得理想结果，国内外在此方面的报道也较少4，这主要是因为传统脱钙方法中，酸性溶液在脱钙的同时会显著降解细胞内的 DNA，进而导致 FISH 检测失败5。本研究经过反复摸索，尝试使用甲酸表面脱钙法对 BMB 组织进行脱钙，提高其石蜡切片荧光原位杂交检测的成功率，为骨髓内淋巴瘤的精确诊治及预后判断提供分子生物学依据。1资料与方法1 1组织来源及分组回顾性收集首都医科大学附属北京友谊医院病理科 2015 年 1 月至 2022 年 5 月的BMB 组织58 例，其中弥漫大 B 细胞淋巴瘤(diffuse largeB cell lymphoma，DLBCL)33 例，套细胞淋巴瘤(mantlecell lymphoma，MCL)21 例，滤泡性细胞淋巴瘤(follicularlymphoma，FL)4 例。根据病理科在不同时期对 BMB 组织常规染色(苏木素和伊红染色、免疫组织化学染色)制片前所用脱钙液及脱钙方法的不同，将实验分为 3 组:硝酸传统脱钙组