Ultrasensitive automated RNA in situ hybridization for kappa and lambda light chain mRNA detects B-cell clonality in tissue biopsies with performance comparable or superior to flow cytometry.
Ultrasensitive automated RNA in situ hybridization for kappa and lambda light chain mRNA detects B-cell clonality in tissue biopsies with performance comparable or superior to flow cytometry.
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DOI：
10.1038/modpathol.2017.142
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发表时间：
2018-03
期刊：
影响因子：
--
通讯作者：
Cook JR
中科院分区：
文献类型：
--
作者：
Guo L;Wang Z;Anderson CM;Doolittle E;Kernag S;Cotta CV;Ondrejka SL;Ma XJ;Cook JR
B细胞克隆性的评估是评估可疑淋巴增殖性疾病的关键组成部分，但如果没有新鲜组织可用于流式细胞仪，从福尔马林固定的石蜡包埋组织中进行分析可能是具有挑战性的。免疫组织化学和常规的Kappa和lambda明场原位杂交染色对浆细胞的评估是有效的，但往往不够敏感，无法检测出B细胞中存在的轻链丰度要低得多的B细胞。我们描述了一种超灵敏的RNA原位杂交分析方法，该方法已被用于自动化免疫组织化学平台，并与流式细胞术在203个连续组织和104个连续骨髓中的结果进行了比较。总体而言，在203例组织活检中，通过流式细胞术，RNA原位杂交发现85例(42%)有轻链限制性B细胞，而58例(29%)。在83例B细胞性非霍奇金淋巴瘤中，流式细胞仪RNA原位杂交发现74例(89%)为限制性B细胞，56例(67%)为限制性B细胞。由于RNA保存不良，仅23/104例(22%)的骨髓可评价B细胞克隆性，但可评价的病例与流式细胞术的符合率为91%。RNA原位杂交可以识别流式细胞仪未发现的双克隆/复合型淋巴瘤，并突出了意想不到的发现，如2例kappa和lambda RNA共同表达，以及1例T淋巴母细胞淋巴瘤中存在lambda轻链RNA。自动RNA原位杂交显示人工评估的观察者间重复性很好(平均K=0.92)，自动图像分析系统与人工评估的符合率高(97%)。自动化RNA原位杂交染色可以在常用的免疫组织化学工具上采用，它允许在福尔马林固定的石蜡包埋组织的形态特征的背景下解释克隆性，其临床敏感性类似于或优于流式细胞术。
The assessment of B-cell clonality is a critical component of the evaluation of suspected lymphoproliferative disorders, but analysis from formalin fixed paraffin embedded tissues can be challenging if fresh tissue is not available for flow cytometry. Immunohistochemical and conventional bright field in situ hybridization stains for kappa and lambda are effective for evaluation of plasma cells, but are often insufficiently sensitive to detect the much lower abundance of light chains present in B cells. We describe an ultrasensitive RNA in situ hybridization assay which has been adapted for use on an automated immunohistochemistry platform and compare results with flow cytometry in 203 consecutive tissues and 104 consecutive bone marrows. Overall, in 203 tissue biopsies, RNA in situ hybridization identified light chain restricted B-cells in 85 (42%) vs. 58 (29%) by flow cytometry. Within 83 B-cell non-Hodgkin lymphomas, RNA in situ hybridization identified a restricted B-cells in 74 (89%) vs. 56 (67%) by flow cytometry. B-cell clonality could be evaluated in only 23/104 (22%) bone marrow cases due to poor RNA preservation, but evaluable cases showed 91% concordance with flow cytometry. RNA in situ hybridization allowed for recognition of biclonal/composite lymphomas not identified by flow cytometry, and highlighted unexpected findings, such as coexpression of kappa and lambda RNA in 2 cases and the presence of lambda light chain RNA in a T lymphoblastic lymphoma. Automated RNA in situ hybridization showed excellent interobserver reproducibility for manual evaluation (average K=0.92), and an automated image analysis system showed high concordance (97%) with manual evaluation. Automated RNA in situ hybridization staining, which can be adopted on commonly utilized immunohistochemistry instruments, allows for the interpretation of clonality in the context of the morphologic features in formalin fixed, paraffin embedded tissues with a clinical sensitivity similar or superior to flow cytometry.
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