Utilization of 1% of Methylene Blue in Staining Histopathological Preparations at Anatomic Pathology Laboratory Tri Rahmawati1, Yadi Apriyadi2, Mamay1 1Department of Medical Laboratory Abstract Technology, STIKes Karsa Husada, Tissue staining using hematoxylin-eosin (HE) is a standard Garut, Indonesia method of histopathological staining. The tissue staining 2Departement Pathology of is hampered when there is no hematoxylin reagent in Anatomi, Regional public hospital laboratory. Therefore, other reagents are needed that can Dr. Slamet, Garut, Indonesia replace the use of hematoxylin. Methylene blue is a basic Correspondence: dyes that interact with cell nuclei which has a negative ionic Mamay, charge of the tissue. It can be used as an alternative nuclei Jl. Nusa Indah No.1, Jayaraga, Kec. staining. This study aims to evaluate the use of 1% of Tarogong Kidul, Kabupaten Garut methylene blue in cell nuclei staining in histopathological Zip Code : 44151 preparations. The research sample were 15 pathology preparations which were randomly selected including breast Email: [email protected] cancer, cervical cancer and ovarian cancer in the bank of sampel at anatomical pathology laboratory of RSUD Dr. Received: May 31, 2020 Slamet Garut, Indonesia. The experiment showed that the Revised: June 19, 2020 methylene blue dyes yielded “worth” result (40%) and Accepted: August 25, 2020 “poorly” result (60%). Further research can be carried out by modifying the pH of 1% of methylene blue reagent so that it can maximize the staining preparations results as good as those using hematoxylin. Keywords Cancer, hematoxylin, histopathology, methylene blue INTRODUCTION prevalence of tumors or cancer in Indonesia Cancer prevalence is increasing in the shows an increase from 1.4 per thousand world every year. According to the populations in 2013 to 1.7 per thousand International Agency for Research on Cancer populations in 2018 (1). In Garut Regency, (IARC), the number of cancer patients especially in the anatomic pathology worldwide increases since 2012. More laboratory of RSUD dr. Slamet Garut, there than14 million new cases of cancer, of which were 189 cancer patients (44.9% of breast 8 million or more cases, died from cancer. cancer patients, 10.5% of cervical cancer, Based on the Ministry of Health of the 8.9% of lymph node cancer, 7.4% of colon Republic of Indonesia data in 2018, the cancer, 5.8% of ovarian cancer, 4.7% of 93 Tri Rahmawati, et al. thyroid cancer, 3.7% of bone cancer, 2.1% of more easily colored into pink with an acidic prostate cancer, 1.05% of parotid cancer staining called acidophilic using a substance (salivary glands), 1.05% of molar pregnancy, eosin color (7). 1.05% of cancer of body fluids, and other The services in the anatomic pathology types of cancers which count up to 9.5%). laboratory are often hampered when the Precancerous and cancer is characterized hematoxylin reagent runs out and it takes a by excessive cell proliferation due to the long time for the reagent to arrive since the increase in mitotic cells (2). The increased availability of hematoxylin is quite rare. and abnormal mitosis indicates a damaged Thus, the process of hematoxylin maturation tissue of an important feature in precancerous requires a long time (8). Nevertheless, the and cancer tests. The identification and examination service in the anatomic quantification of mitotic cells is used in pathology laboratory must be done histological examination to support the continuously. Therefore, other reagents are diagnosis (3). Histological staining has been needed that can replace the use of widely used in pathology (4). Examination of hematoxylin in histopathological the development of cancer can be conducted preparations staining. microscopically through histopathology The other reagents that can be applied for examination. staining are crystals violet (2, 3, 9), toluidine Important stages of histopathological blue, neutral red, methylene green, and examination starts from fixation, processing, methylene blue (4, 10, 11). Methylene blue is embedding, cutting and staining (5). Tissue a cationic dye that has a positive ionic charge staining using the hematoxylin-eosin (HE) is so that it will interact with cell nuclei that a standard method of histopathological have a negative ionic charge of the tissue (4) staining. Hematoxylin is a basic dye that is Löffler's alkaline of methylene blue is used in commonly used for staining cell nuclei and histopathologically conjunctival epithelial provides a bluish tint while eosin yields a cells and blue-colored neutrophils and more pink dye (2). The principle of such coloration intense colored nuclei (12). Previous stain is chemical interactions binding between with 1% of methylene blue detect oral tissue and dyes (4). The tissues with negative dysplasia and carcinoma resulted in good or anionic charges are more easily stained validity (11). However, the utilization of 1% with a blackish-blue color with an alkaline of methylene blue for histopathological staining called basophilic using hematoxylin staining cancer mitosis has not been reported. dyes. Meanwhile, tissue components with a Therefore, the author aims to apply positive or cationic charge (cytoplasm) are methylene blue compound in the process of 94 Ina J Med Lab Sci Tech 2020; 2(1): 93-100 Tri Rahmawati, et al. histopathological preparations as a substitute 70oC for 45 minutes, starting from 70%, 80% for hematoxylin in the hematoxylin-eosin and 95%. The clearing step used xylol I and staining. xylol II, respectively, for 45 minutes in the oven with a temperature of 65oC–70oC. The MATERIALS AND METHODS impregnation step used liquid paraffin for 45 minutes in an oven with a temperature of The tools used in this study were 65oC–70oC. Furthermore, the embedding step microtome knife, pencil, camera, tissue, label was done to plant the tissue into paraffin paper, oven, cover glass, object-glass, beaker molds then it was stored in the refrigerator for glass, microscope, microtome, water bath, 2 hours until it was solidified completely. cassette, tweezers, mold, refrigerator, pipette, Lastly, a gross cut with a thickness of 4 and staining jar. The materials used include microns was done to obtain a network band. tissue cuts, 10% of buffered formalin, The tissue tape was affixed to the glass object absolute alcohol, 96% of alcohol, 80% of and was followed by mounting in a water alcohol, 70% of alcohol, xylol, paraffin, bath with a temperature of 40oC to obtain lithium carbonate, Mayer hematoxylin, eosin, histopathological preparations that were blue methylene, tap water, distilled water, ready to be coloured. and entelan. Histopathological preparations for the This research was conducted in the control group used the Mayer hematoxylin- sitohistotechnology laboratory from July to eosin staining method while the experimental August 2019. Sample were obtained from 15 group used methylene blue-eosin staining. pathology preparations and were randomly The blue methylene used 1% of distilled selected, including breast cancer, cervical water. The results of staining was analysed cancer and ovarian cancer in the bank of under a microscope with a magnification of sampel at anatomical pathology laboratory of 400x. An optimal-quality staining as nuclei RSUD Dr. Slamet Garut, Indonesia. The exhibiting ‘blue hematoxylin’ with quality assessment of the preparation is chromatin patterns, permit the differentiation carried out by an anatomist pathologist. of distinct cell type (13). A sub-optimal The sample was fixed in the second stage staining, the nuclei were weakly stained and with a 10% of formalin buffer solution for 24 appear pale (14). The data were categorized hours. The tissue preparation consisted of based on the quality of the stained nuclei several stages, including dehydration, (Chapman category modified), it is classified clearing, impregnation, embedding and tissue as “good” if the nucleus was clearly stained cutting. The dehydration process used 95 o and the chromatin was clearly visible; alcohol in an oven at a temperature of 65 C– Ina J Med Lab Sci Tech 2020; 2(1): 93-100 Tri Rahmawati, et al. “worth” if the nucleus was stained but less were three lobes in one cell), as shown in clear and the chromatin was lacking clearly figure 1 (a). The results of staining the cell visible, and it was classified as “poorly” if the nucleus with methylene blue-eosin in a breast cell nucleus is not colored. cancer preparation showed poor staining quality that was characterized by colorless RESULTS cell nuclei, mitotic cell nuclei and chromatin The quality of staining cell nuclei using were not observed, as shown in Figure 1 (b). hematoxylin-eosin in five breast cancer Meanwhile, the four breast cancer preparations have a pretty good staining preparations has good quality (the cell nuclei were visible with coarse chromatin). The quality, the cell nucleus was colored but it was not seen and the chromatin was less results of the staining showed the presence of mitotic cell nuclei (observed cell nuclei that clear, as shown in figure 1 (c). a b c Fig 1. Staining cell nuclei in breast cancer preparations with 400x magnification. (a) Hematoxylin, (b–c) methylene blue The quality of staining of cell nuclei with good quality. The cell nucleus was stained hematoxylin-eosin in four cervical cancer but it was not seen and chromatin was less preparations showed good staining quality clear so that there should be a cell nucleus, as with clear observations of cell nuclei. The in figure 2 (b). Meanwhile, the four cervical appearance of mitotic cell nuclei is shown in cancer preparations showed poor quality figure 2 (a). The results of cell nucleus colour which means that the cell nucleus was staining with methylene blue-eosin in a not stained and chromatin was not observed, cervical cancer preparation showed a quite as shown in Figure 2 (c). 96 Ina J Med Lab Sci Tech 2020; 2(1): 93-100 Tri Rahmawati, et al. a b c Fig 2.