Advance Publication Acta Histochem. Cytochem. 44 (3): 133–139, 2011 doi:10.1267/ahc.11006 J01TRAa03oecHp.4kgt1aCa47yu2n-ol1H65aS,17r98ioJ0s/90Aatc0o10phi6recatctihn.yc1el1meo0fi0cH6aisetoCchyetomcihsetrmyiacnad Cytochemistry A Novel Immunohistochemical Staining Method Allows Ultrarapid Detection of Lymph Node Micrometastases While Conserving Antibody Hiroshi Toda1, Yoshihiro Minamiya1, Masami Kagaya2, Hiroshi Nanjo3, Yoichi Akagami2, Hajime Saito1, Manabu Ito1, Hayato Konno1, Satoru Motoyama1 and Junichi Ogawa1 1Department of Thoracic Surgery, Akita University Graduate School of Medicine, 1–1–1 Hondo Akita City 010–8543, Japan, 2Akita Prefectural Research and Development Center, 11–4 Sanuki Araya Akita City 010–1623, Japan and 3Division of Clinical Pathology, Akita University Hospital, 1–1–1 Hondo Akita City 010–8543, Japan 30 Received February 2, 2011; accepted April 18, 2011; published online June 3, 2011 © 201W1eThdeeJvaeplaonpSeodciaetynofveHlisutoltcrahreampiisdtryimanmduCnoy-histochemical staining method in which an AC electric field is used to facilitate detection of tumor cells. Frozen sections of non-small cell lung cancer in lymph nodes were fixed in acetone for 2 min, after which they were incubated for 2 min with an anti-pancytokeratin antibody cocktail and then with EnVisionTM complex under an alternating current (AC) electric field. The sections were then incubated with a chromogen (3,3'diaminobenzidine) for 3 min and counterstained with hematoxylin. This method enabled detection of tumor cells in frozen sections in less than 15 min. In addition, we were able to reduce the amount of antibody used by more than 90% when the sections were incubated under the AC electric field for a longer period. This method could be a useful tool for frozen section diagnosis and research. Furthermore, with this method the cost of immunohistochemical staining can be reduced. Key words: immunohistochemical staining, lymph node metastases, micrometastases I. Introduction Detection of single tumor cells or small tumor cell clusters within lymph nodes and specimens from the sur- gical margin of resected tissue is very important in intra- operative frozen section analysis. In particular, micro- metastases in lymph nodes sometimes cannot be detected on the basis of hematoxylin and eosin (HE) staining alone, but only after additional time-consuming immunohisto- chemical (IHC) investigation of paraffin-embedded tissue. This is noteworthy, as nodal micrometastasis is reportedly associated with poorer survival rates than node-negative disease in breast cancer [4, 22, 25] and non-small cell lung cancer (NSCLC) [5, 10]. Recently, the sentinel node con- cept was introduced into the surgical procedures for breast Correspondence to: Yoshihiro Minamiya, M.D., Ph.D., Associate Professor, Department of Thoracic Surgery, Akita University Graduate School of Medicine, 1–1–1 Hondo Akita City 010–8543, Japan. E-mail: minamiya@med.akita-u.ac.jp cancer [15], malignant melanoma [2], gastric cancer [14] and NSCLC [20]. The rapid, reliable and sensitive intra- operative histopathological evaluation of distant sentinel nodes represents a crucial element of successful applica- tion of the sentinel node concept. However, Turner et al. reported that 10 (14.3%) of 70 patients assessed as tumor- free using routine HE staining were found to be sentinel node-positive using IHC cytokeratin analysis [26]. Unfortu- nately, although the sensitivity of IHC analysis using anti- cytokeratin antibody is sufficient to detect micrometastasis, the standard protocol requires 2–4 hours to complete. To solve this problem, several investigators have proposed rapid methods that enable IHC protocols to be accom- plished within only 12 to 30 min [6, 7, 12, 13, 16–19, 23]. In addition, we have developed a rapid method for detect- ing cytokeratin-positive tumor cells in lymph nodes using flow cytometry [24], which enables us to detect lymph node micrometastases within 40 min. None of these methods is problem free, however. For example, our use of flow cytom- etry was limited by the frequency of false-positives caused © 2011 The Japan Society of Histochemistry and Cytochemistry