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In histological processing, the loss of a small biopsies can prevent diagnosis by the pathologist. Appropriate specimen marking dyes are helpful, but those sold for the purpose have trade-secret components. The purpose of this study is to find suitable dyes with known chemistry to improve the visibility of small specimens. Samples of various organs, including stomach, lung, nasopharynx, small intestine and sentinel lymph nodes, were labeled with Rose red D-FR (CI 282855, Direct red 227), Blue 2RL (CI 24315, Direct blue 80), and Purple D-5BL (CI 29120, Direct violet 66). Clinical pathologists evaluated the dyeing capability and determined any interference of the marking dyes with diagnosis of stained sections. Direct red 227, Direct blue 80, and Direct violet 66 all increased the visibility of small specimens, without interfering with hematoxylin & eosin (HE) staining or immunohistochemistry. All three dyes can therefore be recommended for marking small specimens such as biopsies.

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PURPOSE: Fluorescence-guided surgery using a tumor-specific antibody-dye conjugate is useful in various cancer types. Fluorescence imaging is a valuable tool both intraoperatively and postoperatively for ex vivo imaging. The color of inks used for tumor specimens during ex vivo specimen processing in pathology is an important consideration for fluorescence imaging since the absorption/emission of the dyes may interfere with the fluorescent dye. This study assesses suitable ink colors for use specifically with IRDye800CW fluorescence imaging. PROCEDURES: Eight tissue-marking inks or dyes (TMDs) commonly used for pathological evaluation were assessed. Agarose tissue-mimicking phantoms containing Panitumumab-IRDye800CW were used as an initial model. Mean fluorescence intensity was measured at 800 nm using both Pearl Trilogy as a closed-field fluorescence imaging system and pde-neo II as an open-field fluorescence imaging system before and after TMD application. An in vivo mouse xenograft model using the human head and neck squamous cell carcinoma FaDu cell line was then used in conjunction with TMDs. RESULTS: The retained IRDye800CW fluorescence on Pearl Trilogy was as follows: yellow at 91.0 ± 4.5%, red at 90.6 ± 2.7%, orange at 88.2 ± 2.2%, violet at 56.6 ± 1.1%, lime at 40.9 ± 1.8%, green at 19.3 ± 2.8%, black at 13.3 ± 0.6%, and blue at 8.1 ± 0.2%. The retained IRDye800CW fluorescence on pde-neo II was as follows: yellow at 86.5 ± 6.4%, red at 77.0 ± 6.2%, orange at 76.9 ± 2.8%, lime at 72.5 ± 9.5%, violet at 59.7 ± 0.4%, green at 30.1 ± 6.9%, black at 17.0 ± 2.7%, and blue at 6.7 ± 1.7%. The retained IRDye800CW fluorescence in yellow and blue TMDs was 42.1 ± 14.9% and 0.2 ± 0.2%, respectively in the mouse experiment (p = 0.039). CONCLUSION: Yellow, red, and orange TMDs should be used, and blue and black TMDs should be avoided for evaluating tumor specimens through fluorescence imaging using IRDye800CW.

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INTRODUCTION: Losing of small tissues during tissue preparatory steps may seriously affect pathological diagnostic performance. Using an appropriate tissue marking dye could be an alternative solution. Therefore, the aim of the study was to find a suitable tissue marking dye to enhance the observable ability of various types of small-size tissues during several steps of tissue preparation. MATERIAL AND METHODS: Various small-size samples of various organs and tissues (0.2 to 0.3 cm), including breast, endometrial, and cervical tissue, stomach, small and large intestine, lung, and kidney, were marked with different dyes such as merbromin, hematoxylin, eosin, crystal violet, and alcian blue prior to tissue processing step and their colored-observable ability was evaluated by pathology assistants. Moreover, the diagnostic interfering effect of each tissue marking dye was determined by pathologists. RESULTS: Merbromin, hematoxylin, and alcian blue increased the colored-observable ability of small tissue samples. We suggest using hematoxylin as a tissue marking dye over merbromin and alcian blue because of less toxicity and no interference effect in the step of routine pathological slide examination. CONCLUSIONS: Hematoxylin could be a suitable tissue marking dye for small-size samples and may improve the preanalytical process of tissue preparation in pathological laboratories.

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BACKGROUND: Although histopathological evaluation after endoscopic submucosal dissection (ESD) is critical to assess the accuracy of endoscopic diagnosis, it is still challenging to perform precise endoscopic to pathological evaluation. We evaluated the importance of tissue marking dye (TMD)-targeted marking for post-ESD specimen guided by magnificent endoscope on histopathological accuracy and endoscopic-to-histopathological reconstruction. STUDY DESIGN: A total of 81 specimens resected by ESD [43 without TMD marking (N-TMD group), and 38 specimens with TMD-targeted cancerous areas marking guided by post-procedural magnifying endoscopy on resected specimens (TMD group)] between January 31, 2019, and January 31, 2022 at the Renmin Hospital of Wuhan University were included in the study. The baseline characteristics of patients, discrepancies between endoscopic and histopathological diagnosis, and the impact of TMD on histopathological diagnosis and reconstruction were analyzed. RESULTS: Discrepancies between endoscopic (pre-ESD) and histopathological (post-ESD) diagnosis increased significantly in TMD group (68.4% (26/38) for tumor areas, 26.3% (10/38) for tumor margins, and 26.3% (10/38) for tumor differentiations) when compared with N-TMD group (p < 0.0001). Deeper sections were achieved in all TMD-marked resected lesions and 27.9% (12/43) lesions in the N-TMD group (p < 0.001). More pathological evaluations in TMD group were changed from curative resection to non-curative resection [6/38(15.8%) vs 1/43(2.3%)] compared with N-TMD group (p < 0.0001). TMD-targeted marking also improved the efficiency of histopathological reconstruction on pre-procedural endoscopic images and benefit endoscopists training. CONCLUSION: TMD-targeted labeling on resected specimens could improve precise endoscopic-to-pathological diagnosis, reconstruction by point-to-point marking and benefit endoscopists training.

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Localization of tumors during laparoscopic surgery is generally performed by locally injecting India ink into the submucosal layer of the gastrointestinal tract using endoscopy. However, the location of the tumor is obscured because of the black-stained surgical field and the blurring caused by India ink. To solve this problem, in this study, we developed a tissue-adhesive porphyrin with polycations consisting of quaternary ammonium salt groups. To evaluate the ability of tissue-adhesive porphyrin in vivo, low-molecular-weight hematoporphyrin and tissue-adhesive porphyrin were injected into the anterior wall of the exposed stomach in rats. Local injection of low-molecular-weight hematoporphyrin into the anterior wall of the stomach was not visible even after 1 day because of its rapid diffusion. In contrast, the red fluorescence of the tissue-adhesive porphyrin was visible even after 7 days due to the electrostatic interactions between the positively-charged moieties of the polycation in the tissue-adhesive porphyrin and the negatively-charged molecules in the tissue. In addition, intraperitoneal injection of tissue-adhesive porphyrin in rats did not cause adverse effects such as weight loss, hepatic or renal dysfunction, or organ adhesion in the abdominal cavity. These results indicate that tissue-adhesive porphyrin is a promising fluorescent tissue-marking agent.

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Pathological biopsy protocols require tissue marking dye (TMD) for orientation. In some cases (e.g., close margin), additional immunohistochemical analyses can be necessary. Therefore, the correlation between the applied TMD during macroscopy and the examined TMD during microscopy is crucial for the correct orientation, the residual tumour status and the subsequent therapeutic regime. In this context, our group observed colour changes during routine immunohistochemistry. Tissue specimens were marked with various TMD and processed by two different methods. TMD (blue, red, black, yellow and green) obtained from three different providers (A, B and C, and Whiteout/Tipp-Ex®) were used. Immunohistochemistry was performed manually via stepwise omission of reagents to identify the colour changing mechanism. Blue colour from provider A changed during immunohistochemistry into black, when 3,3'-Diaminobenzidine-tetrahydrochloride-dihydrate (DAB) and H2O2 was applied as an immunoperoxidase-based terminal colour signal. No other applied reagents, nor tissue texture or processing showed any influence on the colour. The remaining colours from provider A and the other colours did not show any changes during immunohistochemistry. Our results demonstrate an interesting and important pitfall in routine immunohistochemistry-based diagnostics that pathologists should be aware of. Furthermore, the chemical rationale behind the observed misleading colour change is discussed.

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Dermoscopy has improved diagnostic precision in the clinical evaluation of complex skin lesions by helping to pinpoint small clinically suspicious areas that are not visible to the naked eye but require histopathologic examination. Derm dotting is a new technique for selectively marking areas of interest to ensure they are examined under a microscope. Left unmarked, suspicious areas might not appear in routinely prepared cross sections or might be difficult to correlate with dermoscopic images. In vivo or ex vivo derm dotting involves the application of nail polish, which, unlike stains, leaves tissues unaltered. Dots can be placed by the dermatologist or laboratory staff during macroscopic preparation of the sample. Derm dotting is a simple, inexpensive technique that any dermatopathology laboratory can quickly adopt to improve diagnostic precision through better dermoscopic-histologic correlation.

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