Goldstein Neal S, Hunter Susan, Forbes Suzy, Odish Eva, Tehrani Matab
Department of Anatomic Pathology, William Beaumont Hospital, Royal Oak, MI 48073, USA.
Appl Immunohistochem Mol Morphol. 2007 Jun;15(2):203-7. doi: 10.1097/01.pai.0000209861.90086.58.
We noticed that the percentage and intensity of estrogen receptor (ER) antibody (Ab) AB ER 1D5 immunohistochemical (IHC) staining was altered by Ab incubation time and the type of chromogen detection system in invasive breast carcinomas. We studied the impact of these 2 factors on Ab ER 1D5 immunoreactivity. Serial sections from 22 strongly ER-positive invasive breast carcinomas were immunohistochemically stained with Ab clone ER 1D5 using 3 IHC protocols. One IHC protocol used a 12-hour Ab incubation and a supersensitive, labeled streptavidin-biotin chromogen detection system (12 h-Standard), the second IHC protocol used a 2-hour Ab incubation and a supersensitive, labeled streptavidin-biotin chromogen detection system (2 h-SS), and the third protocol used a 2-hour Ab incubation and a polymer-based detection system (2 h-Env). Twenty identical fields on each slide stained with each IHC protocol were evaluated and staining was quantified using image analysis. The mean staining percentages using the 12 h-Standard, 2 h-SS, and 2 h-Env IHC staining protocols were 89%, 72%, and 47%, respectively (P<0.001). Three of the 22 cases (14%) were ER negative (<10% stained area) with the 2 h-Env IHC protocol. Stain intensity was significantly stronger with the 12 h-Standard Ab incubation IHC protocol than either 2-hour Ab incubation protocol (P<0.001). Twelve cases stained with 2-hour Ab incubation IHC protocols had weak visually seen staining: 7 were Allred total score 2 (ER negative) and 5 were Allred total score 3. Ab ER 1D5 avidity is directly related to factors that impact electrostatic forces, one of which is Ab incubation time. Standard automated stainer Ab incubation times of less than 1 hour may be of insufficient duration and result in artificially low levels of ER immunoreactivity. The chromogen detection system in association with the ER 1D5 Ab also alters levels of immunoreactivity. Optimization of IHC staining protocols should include evaluating the Ab incubation time and chromogen detection system. These factors can substantially alter the extent and intensity of ER IHC staining.
我们注意到，在浸润性乳腺癌中，雌激素受体（ER）抗体（Ab）AB ER 1D5免疫组化（IHC）染色的百分比和强度会因抗体孵育时间和显色剂检测系统的类型而改变。我们研究了这两个因素对Ab ER 1D5免疫反应性的影响。使用3种免疫组化方案，对22例ER强阳性浸润性乳腺癌的连续切片进行Ab克隆ER 1D5免疫组化染色。一种免疫组化方案采用12小时抗体孵育和超敏的标记链霉亲和素-生物素显色剂检测系统（12小时标准方案），第二种免疫组化方案采用2小时抗体孵育和超敏的标记链霉亲和素-生物素显色剂检测系统（2小时超敏方案），第三种方案采用2小时抗体孵育和基于聚合物的检测系统（2小时增强方案）。对每张玻片上用每种免疫组化方案染色的20个相同视野进行评估，并使用图像分析对染色进行定量。采用12小时标准方案、2小时超敏方案和2小时增强方案免疫组化染色的平均染色百分比分别为89%、72%和47%（P<0.001）。在2小时增强方案免疫组化染色中，22例病例中有3例（14%）ER阴性（染色面积<10%）。12小时标准抗体孵育免疫组化方案的染色强度明显强于两种2小时抗体孵育方案（P<0.001）。采用2小时抗体孵育免疫组化方案染色的12例病例肉眼可见染色较弱：7例为阿尔雷德总分2分（ER阴性），5例为阿尔雷德总分3分。Ab ER 1D5的亲和力与影响静电力的因素直接相关，其中之一是抗体孵育时间。标准自动染色仪的抗体孵育时间少于1小时可能持续时间不足，并导致ER免疫反应性人为降低。与ER 1D5抗体相关的显色剂检测系统也会改变免疫反应性水平。免疫组化染色方案的优化应包括评估抗体孵育时间和显色剂检测系统。这些因素可显著改变ER免疫组化染色的程度和强度。
