Staining Quality Research Articles

Dropper method for cytology slide staining: An effective and sustainable approach to eliminating cross contamination.

Cytopathology laboratories must ensure effective measures to prevent cross-contamination during slide staining. As such, slides with high potential for cross-contamination are usually stained separately, by dipping slides into a series of Romanowsky-type stains, with periodic (usually weekly) filtering and replacement of stains used. Our 5-year experience and a validation study of an alternative dropper method is presented. In this method, cytology slides are placed in a staining rack, a small amount of stain is applied to each using a dropper. Because of the small amount of stain used, this dropper method requires no filtering or reuse of stain, eliminating the possibility of cross contamination and reducing the total amount of stain used. In our 5-year experience, we report a complete elimination of cross contamination due to staining, excellent staining quality, and a slight reduction in overall stain expenditure.

A comparative analysis of two antigen retrieval techniques: Microwave oven and pressure cooker for immunoexpression of estrogen and progesterone receptors in breast cancer tissue

Objectives: Antigen retrieval (AR) is an important step in Immunohistochemistry (IHC) which is used to unmask the antigenic sites and facilitate antigen-antibody binding. Adequate fixation of tissue is necessary to achieve consistent demonstration of tissue antigens that can be masked by the chemical process involved in formalin fixation and tissue processing. Out of the various methods of AR, heat-induced epitope retrieval (HIER) methods have greatly improved the quality and reproducibility of IHC. In this study, a comparison of the two most commonly used HIER methods-pressure cooker and microwave oven was done on thirty cases of breast carcinoma. Materials and Methods: Appropriate tumor sections were taken and subjected to manual IHC testing for estrogen receptor (ER) and progesterone receptor (PR) receptors in each case. The results were divided into technique and microscopy-based. The parameters assessed on microscopy were uniformity of nuclear staining, quality of nuclear staining, internal control staining, presence of background staining, and Allred score. The sensitivity and specificity and positive and negative predictive values for each method were calculated. Results: The parameters assessed on microscopy were comparable for both methods. Using a microwave oven, the sensitivity and specificity for ER and PR were 94% and 100%, respectively. Using a pressure cooker, the sensitivity, and specificity for ER were 94% and 100%, respectively, and for PR were 88% and 100%, respectively. On technical aspects, the pressure cooker method offers the advantage of being more convenient due to the possibility of simultaneous handling of more slides and being more time efficient. Conclusion: Both the AR methods had comparable results on microscopy. However, the pressure cooker has the benefit of being both time and money efficient from a technical standpoint.

Optimizing the Cell Painting assay for image-based profiling.

In image-based profiling, software extracts thousands of morphological features of cells from multi-channel fluorescence microscopy images, yielding single-cell profiles that can be used for basic research and drug discovery. Powerful applications have been proven, including clustering chemical and genetic perturbations on the basis of their similar morphological impact, identifying disease phenotypes by observing differences in profiles between healthy and diseased cells and predicting assay outcomes by using machine learning, among many others. Here, we provide an updated protocol for the most popular assay for image-based profiling, Cell Painting. Introduced in 2013, it uses six stains imaged in five channels and labels eight diverse components of the cell: DNA, cytoplasmic RNA, nucleoli, actin, Golgi apparatus, plasma membrane, endoplasmic reticulum and mitochondria. The original protocol was updated in 2016 on the basis of several years' experience running it at two sites, after optimizing it by visual stain quality. Here, we describe the work of the Joint Undertaking for Morphological Profiling Cell Painting Consortium, to improve upon the assay via quantitative optimization by measuring the assay's ability to detect morphological phenotypes and group similar perturbations together. The assay gives very robust outputs despite various changes to the protocol, and two vendors' dyes work equivalently well. We present Cell Painting version 3, in which some steps are simplified and several stain concentrations can be reduced, saving costs. Cell culture and image acquisition take 1-2 weeks for typically sized batches of ≤20 plates; feature extraction and data analysis take an additional 1-2 weeks.This protocol is an update to Nat. Protoc. 11, 1757-1774 (2016): https://doi.org/10.1038/nprot.2016.105.

Pros and Cons of Air-Dried and Conventional Wet-Fixed Pap Smears: An Institutional Study

Introduction: Wet-fixed smear study routinely used to identify several pre-cancerous and cancerous tumours. By contrasting cellular preservation and staining quality in rehydration smears with wet-fixed smears, the current study aims to establish the effects of rehydration on air-dried smears. Materials and Methods: One hundred paired samples are compared. Wet fixing was done in the first place. Air-dried samples are rehydrated for 30 s with regular saline and then placed in 95% ethanol for 30 min. Both smears will undergo screening, evaluation and grading for numerous cytomorphological factors after Pap staining. The 2014 Bethesda system was used to report the cervical smear results. Results: When evaluated for numerous cellular properties, statistically significant similarities can be seen between two groups, one with wet-fixed smears and the other with rehydrated air-dried smears. The results of the cytological diagnosis comparison between the two groups can be shown in the agreement. Rehydrated air-dried smear interpretation and staining quality are significantly superior to traditional wet smears. Conclusions: Rehydration air-dried techniques can be used instead of traditional wet fixation methods and are as effective when used alone or with traditional wet-fixed smears.

Prolonged Duration of Air-Dry Fixation of Cervical Smears Produces Superior Cytomorphological Staining Quality over Conventional Wet-Fixed Smears

Introduction: Immediate fixation of smears in 95% alcohol for Pap staining is commonly used for cytopathological diagnosis of cancers and other diseases. Few research studies have investigated the comparative outcomes of alcohol wet fixation and rehydration of air-dried smears, indicating that air-dried rehydrated (ARF) smears are viable alternative to wet-fixed (WF) smears. However, there are no or few investigations on the effects of long durations of air-drying fixation on cytomorphological staining quality. Materials and Methods: 124 cervical smears were obtained from the Family Planning Unit of Komfo Anokye Teaching Hospital in Kumasi, Ghana. Quadruple smears were WF and air-dried for 2 h, 4 h, and 8 h prior to rehydration with normal saline and fixation (ARF). All the smears were stained with Papanicolaou stain, examined microscopically for their cytomorphological features, and scored. Cytomorphological scores were statistically analyzed using SPSS software. Results: No significant difference in cytolysis, cell border, nuclear border, chromatin, and cellularity between WF and ARF was observed. However, significant differences in cytoplasmic staining quality (p value <0.001) and the absence of RBCs (p value <0.001) were observed in the 4-h ARF. The absence of RBCs in the ARF smears rendered a clearer background than in the wet fixation. Conclusions: ARF, Pap-stained smears showed comparably superior cytomorphological features to those of WF smears. The 8-h ARF smears produce crispy chromatin and excellent background, making them suitable for bloody cytological samples.

Protecting RNA quality for spatial transcriptomics while improving immunofluorescent staining quality.

In comparison to bulk sequencing or single cell sequencing, spatial transcriptomics preserves the spatial information in tissue slices and can even be mapped to immunofluorescent stainings, allowing translation of gene expression information into their spatial context. This enables to unravel complex interactions of neighboring cells or to link cell morphology to transcriptome data. The 10× Genomics Visium platform offers to combine spatial transcriptomics with immunofluorescent staining of cryo-sectioned tissue slices. We applied this technique to fresh frozen mouse brain slices and developed a protocol that still protects RNA quality while improving buffers for immunofluorescent staining. We investigated the impact of various parameters, including fixation time and buffer composition, on RNA quality and antibody binding. Here, we propose an improved version of the manufacturer protocol, which does not alter RNA quality and facilitates the use of multiple additional antibodies that were not compatible with the manufacturer protocol before. Finally, we discuss the influence of various staining parameters, which contribute to the development of application specific staining protocols.

Standardization of Grocott's methenamine (hexamine) silver method for glycogen demonstration in liver tissue.

Demonstration of glycogen can be done in different lesions and is considered diagnostically significant, mainly in some tumors. Glycogen staining is affected by the type of fixative, the temperature of fixation, and the staining technique.Grocott's methenamine (hexamine) silver technique quality was assessed after four different types of fixatives at two different temperatures [Bouin's solution, 10% neutral buffered formalin (NBF), 80% alcohol, and Rossman's solution at room temperature (RT) and 4°C, for 24h]. These variables were studied to optimize this technique for glycogen demonstration. Archived paraffin blocks were used in this study. They were prepared from one rabbit's liver, and 32 paraffin sections were prepared and stained with Grocott's methenamine (hexamine) silver technique. Eightypercent alcohol provided the highest staining quality scores at both RT and 4°C in comparison with the other fixatives. We concluded that 80% alcohol at 4°C seems to be the fixative of choice for glycogen with the Grocott's methenamine (hexamine) silver technique at the level of this study.

Topological-Preserving Membrane Skeleton Segmentation in Multiplex Immunofluorescence Imaging.

Multiplex immunofluorescence (MxIF) is an emerging imaging technology whose downstream molecular analytics highly rely upon the effectiveness of cell segmentation. In practice, multiple membrane markers (e.g., NaKATPase, PanCK and β-catenin) are employed to stain membranes for different cell types, so as to achieve a more comprehensive cell segmentation since no single marker fits all cell types. However, prevalent watershed-based image processing might yield inferior capability for modeling complicated relationships between markers. For example, some markers can be misleading due to questionable stain quality. In this paper, we propose a deep learning based membrane segmentation method to aggregate complementary information that is uniquely provided by large scale MxIF markers. We aim to segment tubular membrane structure in MxIF data using global (membrane markers z-stack projection image) and local (separate individual markers) information to maximize topology preservation with deep learning. Specifically, we investigate the feasibility of four SOTA 2D deep networks and four volumetric-based loss functions. We conducted a comprehensive ablation study to assess the sensitivity of the proposed method with various combinations of input channels. Beyond using adjusted rand index (ARI) as the evaluation metric, which was inspired by the clDice, we propose a novel volumetric metric that is specific for skeletal structure, denoted as . In total, 80 membrane MxIF images were manually traced for 5-fold cross-validation. Our model outperforms the baseline with a 20.2% and 41.3% increase in and ARI performance, which is significant (p<0.05) using the Wilcoxon signed rank test. Our work explores a promising direction for advancing MxIF imaging cell segmentation with deep learning membrane segmentation. Tools are available at https://github.com/MASILab/MxIF_Membrane_Segmentation.

Abstract 677: Increasing plexity of imaging mass cytometry for tumor tissue analysis

Abstract Imaging Mass Cytometry™ (IMC™) is the leading platform for high-plex tissue imaging. IMC allows for detailed assessment of cell phenotype and function using 40-plus markers simultaneously at subcellular resolution on a single slide. A comprehensive IMC panel containing structural, functional, and immune markers enables us to reveal the complex heterogeneity of tumor tissues as well as the tumor microenvironment (TME). Driven by an increase in the number of antibody markers and the addition of mRNA markers, there is an increasing demand for larger panels. In addition, increasing the number of investigated target markers on a single tissue enriches spatial characterization that may facilitate a more accurate prediction of disease progression and preclinical outcome measures in clinical research projects using tumor biopsies or tissue microarrays (TMAs). Therefore, to increase the plexity of IMC panels, it is essential to expand the number of available metal channels. Here, we demonstrate the incorporation of conjugated antibodies with yttrium (89Y) and indium (115In), two low-mass metals, for IMC application. These metal tags have been previously tested as putative channels for IMC application. We performed IMC analysis of various tissue types stained with panels of conjugated antibodies including the novel 89Y- and 115In-conjugated antibodies. At least 3 different regions of interest (ROIs) were assessed for each of the investigated tissue types. We compared images for the 89Y- and 115In-conjugated antibodies with the images generated using Maxpar® catalog antibodies of the same clones, with a focus on marker specificity and background signal. Compared with the lanthanide-conjugated catalog antibodies, the 89Y- and 115In-conjugated antibodies showed equivalent specificity and staining quality. Our results open a new avenue to assign markers to 89Y and 115In, which enables a larger list of potential targets to be investigated in any IMC study. Expanding the number of markers to 40-plus in Imaging Mass Cytometry will improve the imaging results necessary to identify novel cell signatures (phenotype and interactions) in the TME. Citation Format: Thomas D. Pfister, Shaida Ouladan, Huihui Yao, Daniel Majonis, Christina Loh, Nick Zabinyakov. Increasing plexity of imaging mass cytometry for tumor tissue analysis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 677.

Abstract 5642: Validation of a novel multiplex immuno-fluorescence panel for the spatial analysis of the tumor microenvironment

Abstract IHC counterparts. Subsequently, the panel was transferred on a multi-organ TMA including several tumoral and non-tumoral specimens, showing robust performance across multiple tissue types. The protocol was optimized to achieve high staining quality for all 13 markers in terms of signal specificity Investigation of the tumor microenvironment (TME) by multiplex immunofluorescence (mIF) has accelerated the understanding of the spatial immune context in tumors. mIF has proven to be a powerful technique for the identification of new potential biomarkers and therapeutical targets. Despite increased application of mIF assays to characterize the TME, state-of-the art protocols remain technically challenging. Manual execution and use of dedicated reagents render them lengthy and costly. Moreover, their reproducibility is often questioned together with their transferability between different tissue types. Here, we show the development and validation of an Immuno-Oncology (IO) Core Panel of 13 clinically relevant biomarkers to enable spatial analysis of the immune TME on the COMET™ platform across various tissue types. Formalin-fixed paraffin-embedded human tissue sections from tonsil and a 24-cores multi-organ tissue microarray (TMA) were stained using the IO Core Panel from Lunaphore on the COMET™ platform by fully automated sequential immunofluorescence (seqIF™), which consists of cycles of staining, imaging, and elution. The panel allows for simultaneous detection of CD3, CD4, CD8, CD45, FoxP3, PD1, PD-L1, CD11c, CD20, CD56, CD68, aSMA and Ki-67 by indirect immunofluorescence using unlabeled primary antibodies and Alexa Fluor™ Plus secondary antibodies. The 13-plex IO Core panel was initially developed and validated on tonsil as positive control tissue. To compare immunofluorescent (IF) and immunohistochemistry (IHC) staining patterns, the sections retrieved from COMET™ after seqIF™, were stained by a histology facility with standard IHC established for routine pathological diagnosis. All markers demonstrate accurate detection with specific IF staining, comparably to gold-standard, sensitivity, ratio to background and dynamic range. The repeatability and reproducibility of the automated IO Core Panel on the COMET™ platform was verified by day-to-day tests on one platform and tests among multiple platforms, respectively. Our study demonstrated the robustness of the validated IO Core Panel across multiple tissue types with highly specific and reproducible results. The marker detection with standard indirect immunofluorescence on the COMET™ instrument allows for future, rapid expansion and customization of the panel including additional primary antibodies towards the need of the individual underlying scientific question. Citation Format: François Rivest, Victor de Gautard, Vytautas Navikas, Saska Brajkovic, Bastian Nicolai. Validation of a novel multiplex immuno-fluorescence panel for the spatial analysis of the tumor microenvironment. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 5642.

Evaluation of biosafe alternative to eosin in hematoxylin and eosin staining procedure: A comparative study.

Hematoxylin and eosin staining is the globally accepted staining technique for histology and histopathology sections. Hematoxylin is a natural dye as compared to eosin which is a synthetic dye manufactured from chemicals. Eosin acts as a counterstain to hematoxylin giving a sharp contrast to its blue color. Eosin being a chemical, can cause health hazards and environmental pollution. Using eco-friendly materials and going organic is the demand of this era. Curcuma longa rhizomes are proven to contain curcuminoids that exhibit various medicinal properties and curcumin the bright yellow component of the plant, that is, being used as a colorant. This natural dye from C. longa provides a significant alternative to eosin as they are safer to use without health hazards and are biodegradable and have easy disposability. To evaluate and compare the staining qualities of C. longa (turmeric) with hematoxylin and eosin (H&E). A total of 100 paraffin-embedded tissue block specimens of various pathological tissues were stained with H and E and hematoxylin and curcumin (H&C). Evaluation and comparison of the staining efficacy were done for all the pathological tissues. The obtained data were subjected to statistical analysis. H&C staining gave comparable results with H and E staining with a statistically insignificant P value (p > 0.05). Curcumin has shown equivalent staining to that of eosin. Thus, it can be used as a better alternative.

A practical protocol to prepare paraffin-embedded whole tick histology sections

Ticks are important ectoparasites that are capable of transmitting multiple classes of pathogens and are currently linked with many emerging tick-borne diseases worldwide. With increasing occurrences of tick-borne diseases in both humans and veterinary species, there is a continuous need to further our understanding of ticks and the pathogens they transmit. Whole tick histology provides a full scope of the tick internal anatomy, allowing researchers to examine multiple organs of interest in a single section. This is in contrast to other techniques that are more commonly utilized in tick-borne disease research, such as electron microscopy and light microscopy of individual organs. There is a lack of literature describing a practical technique to process whole tick histologic sections. Therefore, the current study aims to provide researchers with a workable protocol to prepare high quality paraffin-embedded whole tick histology sections. Amblyomma americanum adults were used as an example species for this study. After a series of pilot experiments using a combination of various fixatives, softening agents and processing techniques, we elected to compare two common fixatives, 10% neutral-buffered formalin (NBF) and Bouin's solution for whole ticks. Equal numbers of A. americanum unfed adults (n = 10/fixative) were processed identically and their whole tick histology coronal sections were individually scored. Higher scores were assigned to whole tick sections that contained more internal organs that are crucial for tick-borne disease research (e.g. salivary glands and midgut), high integrity of tissues and exoskeleton on the section, and good fixation and staining quality of the tissues. The mean total scores for Bouin's-fixed ticks were significantly higher compared to NBF-fixed ticks (p = 0.001). To further assess our preferred technique, we also demonstrated the feasibility of producing high quality whole tick sections for three other common tick species of medical importance (Rhipicephalus sanguineus, Ixodes scapularis, and Dermacentor variabilis) using Bouin's solution. While this technique may require further optimization for other tick species, we described a feasible protocol that uses commonly available tools, reagents and standard histologic equipment. This should allow any investigator to easily make adjustments to this protocol as needed based on their experimental goals.

Role of dry ice in decoverslipping of microscopic slides: A new insight.

The process of decoverslipping is often required in a laboratory to review or examine an older slide which tends to fade over time, making it almost impossible to use it for research or study purposes. The sections then need to be re-stained which can only be done after removing the coverslip. The traditional method of decoverslipping using xylene is a time-consuming process. Various methods have been used in the past; however, none were found to be completely effective. Dry ice, the solid form of carbon dioxide, is an easily available, cheap cooling agent with a low freezing temperature (-78.5°C) which was evaluated for its efficacy in decoverslipping process, as an alternative to xylene. 64 faded haematoxylin and eosin (H&E)-stained histopathology slides were randomly selected and segregated, according to duration of year, into eight major groups. Each group was further divided into four subgroups according to the time that the slides were subjected for decoverslipping. The slides were placed on dry ice and the time was set. Once the coverslip was removed, the slides were placed in xylene to remove any residual mountant. The tissue sections were evaluated for physical disfigurement followed by re-staining with H&E to check for any change in tissue morphology. The mean time taken for removal of coverslip using dry ice was 35 seconds. This technique is easy, fast, and effective, with no tissue loss or compromise in staining quality, thereby preventing xylene toxicity and its effect on the environment.

Hibiscus Sabdariffa as Nuclear Stain for Cytological Buccal and Cervical Smear

Introduction: Sudan is one of the biggest countries that produces and exports hibiscus Sabdariffa in Africa. The possibility of HS staining cytological specimens is not well explored. Therefore, the present study aimed to evaluate the staining quality of Hibiscus Sabdariffa on cytological buccal and cervical smear as a cheap viable nuclear stain alternative to hematoxylin. Methods: This study was an experimental descriptive study conducted at Alfajr College for Science and Technology from (June to September 2022). A total of 30 smears, 15 buccal smears collected from students and 15 cervical smears were retrieved from Alfajr’s histopathology lab. Results: All (30) slides showed good staining quality with 20% concentration Hibiscus Sabdariffa water extract when mordanted with Iron, and acidified with acetic acid. Conclusion: Hibiscus Sabdariffa is good as nuclear staining for a buccal and cervical pap smear and can replace hematoxylin by adjustment of concentration, time and pH.

Efficacy of Kumkum as a Surrogate for Eosin in Routine Histological Sections: An Observational Study

Introduction: Stains are a crucial component of laboratory procedure. They help in highlighting the different tissues, both normal and abnormal, which plays an important role in diagnostic histopathology. Synthetic dyes are utilised for majority of the stains in histology. Among the natural surrogates studied, Kumkum is less researched. Aim: To compare the staining characteristics of Kumkum, which imparts red colour as an alternative to eosin. Materials and Methods: This observational study was conducted at Histopathology section, Pathology department at Chettinad Hospital and Research Institute for a period of six months from January 2022 to June 2022. In 50 tissue blocks, each was made into two sections and one was stained with routine Haematoxylin and Eosin (H&amp;E), while the other was stained with Haematoxylin and Kumkum solution (H&amp;K). The Cellular Architecture (CA) (based on distinct or indistinct nucleus, cytoplasm) and quality of staining (poor, satisfactory or good) of H&amp;E and H&amp;K solution was compared and a scoring system was given. The scores were analysed with Chi-square test using SPSS software (version 25). Results: Out of 100 slides, 45 (90%) stained with H&amp;E and 48 (96%) with H&amp;K showed distinct CA. A total of 47 slides (94%) stained with H&amp;K and 41 (82%) with H&amp;E showed good quality of staining. Conclusion: Kumkum solution appears to be an efficient counterstain in place of eosin in highlighting the normal structures in histopathological sections. It highlighted the RBCs in the arteries and cytoplasm of the cells in glandular and squamous epithelium better than or equally good as eosin.

Visualization and quality control tools for large-scale multiplex tissue analysis in TissUUmaps3.

Large-scale multiplex tissue analysis aims to understand processes such as development and tumor formation by studying the occurrence and interaction of cells in local environments in, for example, tissue samples from patient cohorts. A typical procedure in the analysis is to delineate individual cells, classify them into cell types, and analyze their spatial relationships. All steps come with a number of challenges, and to address them and identify the bottlenecks of the analysis, it is necessary to include quality control tools in the analysis workflow. This makes it possible to optimize the steps and adjust settings in order to get better and more precise results. Additionally, the development of automated approaches for tissue analysis requires visual verification to reduce skepticism with regard to the accuracy of the results. Quality control tools could be used to build users' trust in automated approaches. In this paper, we present three plugins for visualization and quality control in large-scale multiplex tissue analysis of microscopy images. The first plugin focuses on the quality of cell staining, the second one was made for interactive evaluation and comparison of different cell classification results, and the third one serves for reviewing interactions of different cell types.

Evaluation of a New Mordant Based Haematoxylin Dye (Haematoxylin X) for Use in Clinical Pathology.

Recently, St John's Dermatopathology Laboratory and CellPath Ltd have developed a new patented haematoxylin dye (Haematoxylin X) that utilises a chromium-based mordant (Chromium Sulphate). In this study, the performance of this new haematoxylin (Haematoxylin X) was compared against some commonly utilised alum-based haematoxylins (Carazzi's, Harris' and Mayer's) when used as a part of formalin-fixed paraffin embedded (FFPE) tissue, special stains, immunohistochemical counterstaining and frozen section (Mohs procedure) staining procedures. FFPE sections of different tissue types and frozen skin tissues were sectioned and stained with each haematoxylin subtype to allow for a direct comparison of staining quality. The slides were independently evaluated microscopically by two assessors. A combined score was generated to determine the sensitivity (defined as the intensity of haematoxylin staining being too weak or too strong and the colour of the haematoxylin staining not being blue/black) and specificity (defined as the presence of haematoxylin background staining, uneven staining, and staining deposits) for each of the four haematoxylin subtypes. The scoring criteria were based on the UKNEQAS Cellular pathology techniques assessment criteria. In FFPE tissue, the results for specificity identified Harris haematoxylin scoring the highest (91.2%) followed by Haematoxylin X (88.0%) and Mayer's (87.0%). The sensitivity scores again identified Harris haematoxylin as scoring the highest (95.1%) followed by Haematoxylin X (90.0%) and Mayer's (88.0%). In frozen tissue, the results for specificity identified Haematoxylin X as scoring the highest (85.5%) followed by Carazzi's (80.7%) and Harris' (77.4%). The sensitivity scores again identified Haematoxylin X as scoring the highest (86.8%) followed by Carazzi's (82.0%) and Harris' (81.0%). The results achieved with all four haematoxylins showed a high degree of comparability, with Harris' haematoxylin scoring high scores overall compared to the other four when assessing FFPE sections. This may have been due to familiarity with the use of Harris' haematoxylin in-house. There was also evidence of more pronounced staining of extracellular mucin proteins with Haematoxylin X compared to the other alum haematoxylins that were assessed. Haematoxylin X scored highest when used in frozen section staining. In addition, Haematoxylin X has a potential applications for use in IHC and special stains procedures as a counterstain.

Image analysis comparison of nerve staining by use of methylene blue, tissue marker, or food dye

Introduction: Novel locoregional techniques require initial dye studies to confirm successful nerve targeting. Historically, these dyes have been subjectively chosen and staining grossly evaluated. The goal of this study was to objectively quantify and compare nerve staining quality using color saturation of dyes commonly reported in the literature.

Determination of reticular fibers in tissues fixed with sugarcane molasses

Reticular fiber consists of one or more types of very thin and delicately woven type III collagen strands, which form a highly ordered cellular network and provide a supportive network. Since most of these collagen types are combined with carbohydrates, they react with silver stains and periodic acid-Schiff reagent, but are not shown by ordinary histological stains like those using hematoxylin. In this study, it was aimed to determine the reticular fibers in tissues fixed with sugarcane molasses and formol using silver staining technique (Gordon Sweet –GS-). The reticular fibers in the tissues fixed with sugarcane molasses were compared with those in the tissues fixed with formol. According to the findings, the staining quality of the liver and spleen tissues fixed with sugarcane molasses showed similar characteristics with the tissues fixed with formol. Very weak staining was observed in kidney and testicle tissues. The fact that the tissues fixed in the same fixation solution show different results in the Gordon Sweet (GS) staining method shows us that this issue needs to be supported by more detailed studies.

Comprehensive optimization of urinary exfoliated tumor cells tests in bladder cancer with a promising microfluidic platform.

Enrichment of urinary exfoliated tumor cells (UETCs) is a noninvasive way of bladder cancer diagnosis, but the lack of specific capture and identification of tumor cells from the urine remains a limitation that impedes the development of liquid biopsy. The CytoBot® 2000, a novel circulating cell isolation and enrichment platform, was used for UETCs isolation after comprehensive optimization. The commercial cell lines of bladder cancer were used in spiking assay for cell recovery test. The flow cytometry and immunofluorescent staining assays were performed for expression validation of capture target and identification markers. The performance of optimized platform was validated by 159 clinical samples and analyzed using receiver operator characteristic curve. The chip that had a pore diameter of 15*20 μm could reduce the background residues while maintaining a higher cell recovery rate. We found that the cell capture ability of chip significantly improved after anti-EpCam antibody encapsulation, but not with T4L6FM1. In identification system optimization, the spiking assay and validation of clinical sample showed that the performance of CK20 and DBC-1 were better that pan-CK in tumor cell identification, in addition, the staining quality is more legible with CK20. The optimized capture chip is more specific for UETCs isolation. CK20 and DBC-1 are both sensitive biomarkers of UETCs in bladder cancer diagnosis. The performance of this optimized platform is excellent in clinical test that improves the accuracy of urine cell testing and provides a new alternative for the clinical application of BLCA liquid biopsy assessment.