Varma M, Linden M D, Amin M B
Department of Pathology, Henry Ford Hospital, Detroit, Michigan 48202, USA.
Mod Pathol. 1999 May;12(5):472-8.
Basal cell-specific, anti-high molecular weight cytokeratin (HMCK) antibody is often used to confirm the diagnosis of prostatic adenocarcinoma, particularly if limited amounts of tissue are available. HMCK is formalin sensitive and requires pretreatment by enzymes or heat if formalin-based fixatives are used. To date, the effect of prolonged formalin fixation on HMCK immunoreactivity has not been systematically studied; this is critical, because the diagnosis of malignancy is based on a negative immunoreaction. In this study, 5 tissue blocks obtained from each of 10 radical prostatectomy specimens were fixed in formalin from 6 hours to 1 month. HMCK immunostaining was performed with monoclonal antibody clone 34betaE12 after pretreatment of the sections by either enzymatic predigestion with pepsin, heat-induced epitope retrieval (HIER) with a microwave, or HIER with a hot plate. For scoring, the staining intensity at 6 hours of formalin fixation was considered as the baseline for that particular antigen retrieval technique. After pepsin predigestion or microwaving, there was progressive loss of HMCK immunoreactivity from 1 week or longer of formalin fixation. HIER with a hot plate yielded consistent results with no decrease in HMCK immunoreactivity with as long as 1 month of formalin fixation. The staining intensity was consistently stronger at all periods of formalin fixation when the hot plate method was used, compared with pepsin predigestion or microwaving. Generally weak HMCK positivity was observed in rare neoplastic cells of 3 of 10 specimens after hot plate HIER but not with pepsin predigestion or microwave antigen retrieval. This sporadic immunostaining of malignant cells was quantitatively and qualitatively distinct from the pattern seen in benign epithelium. We conclude that formalin fixation affects HMCK immunoreactivity over time and might impact its diagnostic usefulness. Efficacies of different antigen unmasking/epitope retrieval techniques vary and must be standardized for individual laboratories.
基底细胞特异性抗高分子量细胞角蛋白（HMCK）抗体常用于确诊前列腺腺癌，尤其是在组织样本量有限的情况下。HMCK对福尔马林敏感，如果使用基于福尔马林的固定剂，则需要通过酶或加热进行预处理。迄今为止，尚未对长时间福尔马林固定对HMCK免疫反应性的影响进行系统研究；这一点至关重要，因为恶性肿瘤的诊断基于阴性免疫反应。在本研究中，从10例根治性前列腺切除标本中各获取5个组织块，用福尔马林固定6小时至1个月。在用胃蛋白酶进行酶消化预处理、用微波进行热诱导抗原修复（HIER）或用热板进行HIER后，用单克隆抗体克隆34betaE12进行HMCK免疫染色。为了评分，将福尔马林固定6小时时的染色强度视为该特定抗原修复技术的基线。经过胃蛋白酶消化或微波处理后，福尔马林固定1周或更长时间后，HMCK免疫反应性逐渐丧失。用热板进行HIER产生了一致的结果，福尔马林固定长达1个月时，HMCK免疫反应性没有降低。与胃蛋白酶消化或微波处理相比，使用热板法时，在福尔马林固定的所有时间段内染色强度始终更强。在热板HIER后，10个标本中有3个标本的罕见肿瘤细胞中普遍观察到较弱的HMCK阳性，但胃蛋白酶消化或微波抗原修复则未观察到。恶性细胞的这种散在免疫染色在数量和质量上与良性上皮细胞中的模式不同。我们得出结论，福尔马林固定会随着时间影响HMCK免疫反应性，并可能影响其诊断效用。不同的抗原暴露/抗原修复技术的效果各不相同，必须为各个实验室进行标准化。
