Havelund Birgitte M, Olsen Dorte A, Andersen Rikke F, Spindler Karen-Lise G, Brandslund Ivan, Jakobsen Anders, Soerensen Flemming B
Department of Oncology, Vejle Hospital, Vejle, Denmark.
Appl Immunohistochem Mol Morphol. 2013 Jul;21(4):298-307. doi: 10.1097/PAI.0b013e31826f4475.
The aim of the present study was to investigate the influence of fixation delay and the perioperative ischemia on hypoxia inducible factor (HIF)-1α gene expression, HIF-1α protein expression, and immunohistochemical (IHC) expression of HIF-1α, GLUT-1, Bcl-2, and Ki-67 in colorectal cancer. The study included 25 surgically removed colorectal tumors. Three sets of samples were collected readily after removal and exposed to 0, 30, and 60 minutes of delay of fixation or freezing. The perioperative ischemia time was registered. In each set of the samples, HIF-1α gene expression was analyzed by quantitative real time polymerase chain reaction, protein concentration of HIF-1α was assessed by enzyme-linked immunosorbent assay, and IHC staining of HIF-1α, GLUT-1, Bcl-2, and Ki-67 was performed. Preoperative formalin-fixed paraffin-embedded biopsies and whole sections of the entire tumor were analyzed by IHC. We found that the HIF-1α gene expression, HIF-1α protein concentration, and IHC expression of HIF-1α, GLUT-1, Ki-67, and Bcl-2 were not systematically affected by either the fixation or freezing delay of the tissue, the perioperative ischemia time, or the total ischemia time (perioperative ischemia+delay of fixation or freezing) in colorectal tumors. However, the intraindividual variation was quite large, which may question the use of individually, non-standardized-handled single biopsies or small tissue samples for analysis of often rather heterogenously expressed biomarkers.
本研究的目的是探讨固定延迟和围手术期缺血对结直肠癌中缺氧诱导因子（HIF）-1α基因表达、HIF-1α蛋白表达以及HIF-1α、葡萄糖转运蛋白1（GLUT-1）、Bcl-2和Ki-67免疫组化（IHC）表达的影响。该研究纳入了25例手术切除的结直肠肿瘤。切除后立即收集三组样本，并分别暴露于固定延迟或冷冻0、30和60分钟的条件下。记录围手术期缺血时间。在每组样本中，通过定量实时聚合酶链反应分析HIF-1α基因表达，采用酶联免疫吸附测定法评估HIF-1α蛋白浓度，并对HIF-1α、GLUT-1、Bcl-2和Ki-67进行IHC染色。通过IHC分析术前福尔马林固定石蜡包埋活检组织和整个肿瘤的全层切片。我们发现，结直肠肿瘤中HIF-1α基因表达、HIF-1α蛋白浓度以及HIF-1α、GLUT-1、Ki-67和Bcl-2的IHC表达均未受到组织固定或冷冻延迟、围手术期缺血时间或总缺血时间（围手术期缺血+固定或冷冻延迟）的系统性影响。然而，个体内差异相当大，这可能会对使用个体的、未经标准化处理的单个活检组织或小组织样本分析通常表达相当异质性的生物标志物的做法提出质疑。
