Ultrarapid IHC to Detect Micrometastases 137 Fig. 3. Detection of micrometastasis using the rapid AC electric field IHC procedures. To confirm the ability of the rapid AC elec- tric field IHC procedure to detect lymph node micrometastasis, we immunohistochemically stained lymph nodes positive for NSCLC micrometastasis with anti-pancytokeratin antibody (AE1/AE3). Panels A and B show low and high magnification images using the rapid AC electric field IHC procedures, respectively. Endogenous peroxidase-positive granulocytes were often observed. However, cytokeratin-positive cancer cell nests could be readily discerned based on their morphology. Bars=1,000 µm (panel A), 500 µm (panel B). IV. Discussion In the present study we demonstrated that tumor cells in lymph nodes can be detected within 11 min under a high voltage, low frequency AC electric field using the device developed at our institute. Furthermore, not only does this method enable rapid IHC staining, it can also potentially reduce the cost of the IHC procedure. Conventional analysis of frozen tissue sections using HE staining enables diagnosis within 20 min at most institutions [27], but the sensitivity of this method for detecting micrometastases is comparatively low. By con- trast, the sensitivity of IHC analysis using anti-cytokeratin antibody is sufficient to detect micrometastasis, but the standard IHC procedure commonly requires 2–4 hr to com- plete. To address this issue, several investigators proposed rapid methods that enable the IHC procedure to be accom- plished within only 12 to 30 min [6, 7, 13, 16–18, 23, 24, 27]; however, these methods are not problem-free. For example, the new EnVisionTM system has been described as a very sensitive and rapid detection system for IHC [24, 27], and Kämmerer et al. and Mönig et al. reduced the time for immunostaining of frozen sections to less than 13 min using the EnVision system. To do so, however, they applied a higher concentration of primary antibody than is used for standard IHC [13, 17]. By contrast, our ultrarapid AC electric field IHC procedure required about the same amount of time as the EnVisionTM system, and we were able to use the same concentration of primary antibody used in the standard IHC procedure. Given the expense of primary antibodies, we believe this represents a significant advantage over other procedures. It is also known that microwave irradiation shortens IHC times. Hatta et al. reported that IHC could be completed within 15 min using an intermittent microwave irradiation method in combina- tion with a prepared immune complex consisting of the primary antibody, secondary antibody and EnVisionTM- solution [11]. The utility of our AC electric field method is comparable to this microwave approach. The cost of IHC analysis is high because primary antibodies are expensive. Therefore, reducing the amount of primary antibody needed for IHC can have a significant impact on cost. In particular, diagnosis of lymph node micrometastasis requires the use of step sections of the lymph nodes in question. According to the 6th edition of cancer staging manual of the American Joint Committee on Cancer, micrometastases are defined as being >0.2 mm but ≤2.0 mm in diameter [9]. Consequently, step sections must be collected every 0.2 mm so as not to miss any tumor tissue. In an effort to reduce the amount of primary antibody needed, we applied the AC electric field IHC procedure. We found that when the AC electric field was applied to sections for 180 min, the concentration of primary antibody could be reduced by more than 90%. The mechanism by which our method promotes the antigen-antibody reaction is not fully understood. In this study we applied a high voltage (3.4 kV, offset 2.4 kV), low frequency (18 Hz) AC electric field to IHC. Before we adopted this approach, we tested a variety of other methods for stirring small amounts of solution, ultimately determining that application of an AC electric field worked best under our conditions. It is known that when an AC electric field is applied to a solution, electro-osmotic vortices are produced [3], which were used by Wang et al. [28] to induce non-contact mixing. However, we do not believe such vortices are responsible for the enhanced antigen-antibody reaction seen in the present study. We previously showed that an interaction force affecting a droplet can be generated as a coulomb force applied from an AC electric field [1]. The generated force reflects the difference between dielectric constants and the electric conductivity. In particular, when a droplet is attracted to