Tissue Preservation: Methods, Quality, Challenges

Liyana Rahman^*
*Correspondence: Liyana Rahman, Departmentof Diagnostic Surgical Morphology, Sabah Institute of Medical Sciences, Kota, Kinabalu, Malaysia, Email:

Introduction

Precise fixation and embedding methods are critically important for achieving high-resolution three-dimensional imaging of delicate biological tissues, such as Drosophila. Optimizing these initial steps is essential for preserving the intricate ultrastructure, which directly enables detailed cellular and subcellular analysis using advanced microscopy techniques. This foundational work ensures the reliability and accuracy of subsequent scientific investigations. [1] The integrity of RNA in tissue samples presents a significant challenge in molecular pathology. A comparative study using RNA sequencing investigated the differences in RNA quality between fresh frozen and formalin-fixed paraffin-embedded (FFPE) tissue samples. This research critically highlights the difficulties in maintaining high RNA quality in FFPE tissues, providing invaluable insights that are vital for genetic research and other molecular analyses where RNA quality is paramount. [2] Standardized protocols are cornerstones for consistent and reliable diagnostic outcomes. Practical guidelines have been established for formalin fixation and tissue processing, specifically tailored for diagnostic immunohistochemistry. These guidelines underscore the absolute necessity of standardized procedures to achieve consistent, high-quality results, which are fundamentally vital for accurate pathological diagnosis and patient care. [3] The search for improved fixative solutions is ongoing, driven by the need for enhanced tissue preservation and reduced toxicity. A study evaluated various fixative solutions for their efficacy in preserving liver and spleen tissues for histological examination. This comparative analysis aimed to identify alternatives to traditional formalin that are either less toxic or more effective in maintaining cellular architecture and antigenicity, contributing to safer and more precise histological studies. [4] Understanding the impact of pre-analytical variables on tissue quality is crucial, especially in sensitive areas like neurodegenerative disease research. Research has extensively explored how post-mortem delay and different fixation methods influence human brain tissue morphology and protein expression. Recognizing and managing these variables is paramount for ensuring tissue integrity, which is a prerequisite for generating reliable scientific findings in neurological studies. [5] Beyond chemical fixation, cryofixation techniques offer a powerful alternative for preparing biological samples, particularly for electron microscopy. This method involves rapid freezing, which is known to preserve cellular structures in a state much closer to their native form compared to chemical fixation. This approach effectively avoids common artifacts introduced by chemical fixatives and dehydrating agents, leading to more accurate ultrastructural observations. [6] High-dimensional immunophenotyping, often conducted using flow cytometry, requires meticulous cell preparation and fixation. A standardized workflow has been outlined to ensure optimal cell viability and antigen preservation. These techniques are indispensable for accurate analysis of complex immune cell populations, allowing researchers to gather reliable data on cellular phenotypes and functions in various biological contexts. [7] Artifacts in histology can significantly impede accurate diagnosis and research interpretation. A comprehensive review systematically details various artifacts encountered in histology, with a particular focus on those arising from improper fixation. This resource is invaluable for diagnosticians and researchers, empowering them to identify and proactively avoid these artifacts, thereby ensuring the accurate interpretation of tissue samples and preventing misdiagnoses. [8] Infectious disease diagnostics critically depend on optimized protocols for microbial detection. A specific study investigated different fixation protocols for detecting Chlamydia trachomatis elementary bodies in both cell cultures and tissue samples. The optimization of fixation methods in this context is absolutely crucial, as it ensures that microbial structures are properly preserved, which is fundamental for accurate identification and subsequent analysis of pathogens. [9] Technological advancements are continuously improving pathology practices. An updated review provides insights into microwave-assisted tissue processing and fixation, highlighting its advantages in significantly accelerating laboratory procedures. This innovative technology can improve diagnostic turnaround times while diligently maintaining tissue quality, representing a notable advancement that enhances efficiency and reliability in pathology laboratories. [10]

Description

The foundational aspect of biological and medical research often hinges on the quality of tissue samples, making optimal fixation and embedding methods indispensable. For instance, achieving high-resolution three-dimensional imaging of delicate structures like Drosophila tissues demands precise techniques to preserve ultrastructure for detailed cellular and subcellular analysis [1]. However, maintaining sample integrity is not always straightforward. A significant challenge lies in preserving biomolecules; studies comparing RNA integrity in fresh frozen versus formalin-fixed paraffin-embedded (FFPE) tissues, through RNA sequencing, underscore the inherent difficulties of retaining high RNA quality in FFPE samples for molecular analyses [2]. This issue is further complicated by the prevalence of histological artifacts, many of which stem directly from improper fixation techniques. A comprehensive review serves as a vital resource for recognizing and preventing these artifacts, ensuring accurate interpretation of tissue samples in diagnostics and research [8].

Recognizing these challenges, the development and adherence to standardized protocols are critically important. Practical guidelines for formalin fixation and tissue processing have been meticulously crafted for diagnostic immunohistochemistry, emphasizing their role in achieving consistent, high-quality results essential for accurate pathological diagnoses [3]. Simultaneously, research actively seeks to innovate beyond traditional methods. Comparative studies evaluate various fixative solutions for tissues like the liver and spleen, aiming to identify less toxic or more effective alternatives to conventional formalin. These efforts prioritize maintaining cellular architecture and antigenicity, seeking to enhance both the safety and efficacy of histological examination methods [4].

The influence of preparation techniques extends profoundly into specialized research areas. For human brain tissue, variables such as post-mortem delay and the choice of fixation methods are known to significantly affect morphology and protein expression. Understanding and controlling these factors is paramount for neurodegenerative disease research, where tissue integrity is a prerequisite for reliable scientific findings [5]. Similarly, in infectious disease diagnostics, optimizing fixation protocols is absolutely crucial. Investigations into detecting Chlamydia trachomatis elementary bodies in various sample types highlight how proper preservation of microbial structures directly enables accurate identification and analysis, underscoring fixation's critical role in public health [9].

Modern advancements in tissue processing encompass both specialized preservation methods and workflow optimizations. Cryofixation, for instance, provides a superior alternative for electron microscopy, allowing biological samples to be rapidly frozen. This technique preserves cellular structures in a near-native state, effectively bypassing artifacts commonly introduced by chemical fixatives and dehydrating agents [6]. Parallel to this, robust and standardized workflows are essential for high-dimensional immunophenotyping using techniques like flow cytometry. These workflows, including meticulous cell preparation and fixation, are indispensable for ensuring cell viability and antigen preservation, crucial for accurate analysis of complex immune cell populations [7]. Furthermore, technological innovations such as microwave-assisted tissue processing and fixation are significantly accelerating laboratory procedures. This approach not only improves diagnostic turnaround times but also diligently maintains tissue quality, marking a significant advancement in routine pathology practices by boosting efficiency without compromising integrity [10].

Conclusion

Optimal tissue preservation is paramount across biological and medical research, underpinning high-resolution imaging and precise molecular analyses. Studies continually refine fixation and embedding methods to maintain delicate ultrastructures, crucial for detailed cellular and subcellular analysis, as shown with Drosophila tissues. The integrity of biomolecules, particularly RNA, presents a challenge; comparisons between fresh frozen and formalin-fixed paraffin-embedded (FFPE) samples reveal significant differences, emphasizing the difficulties in maintaining high RNA quality in FFPE for subsequent genetic research. Standardized protocols for formalin fixation and tissue processing are indispensable, especially for diagnostic immunohistochemistry. These guidelines aim to ensure consistent, high-quality results, which are vital for accurate pathological diagnoses. Researchers also actively evaluate various fixative solutions for preserving tissues like liver and spleen, seeking alternatives to traditional formalin that are both less toxic and more effective in maintaining cellular architecture and antigenicity. The impact of pre-analytical variables, such as post-mortem delay and specific fixation methods, significantly influences human brain tissue morphology and protein expression. Understanding these variables is critical for neurodegenerative disease research, ensuring tissue integrity for reliable scientific findings. Complementing chemical approaches, cryofixation techniques offer a method for preparing biological samples for electron microscopy, preserving cellular structures closer to their native state and circumventing artifacts commonly introduced by chemical fixatives. Beyond specific applications, the broader field emphasizes standardized workflows for cell preparation and fixation in high-dimensional immunophenotyping, particularly for flow cytometry. This ensures cell viability and antigen preservation, which are indispensable for accurate analysis of complex immune cell populations. Furthermore, systematic reviews detail various artifacts encountered in histology, frequently stemming from improper fixation, serving as essential resources for diagnosticians to identify and avoid errors. The development of optimized fixation protocols is also crucial for infectious disease diagnostics, ensuring microbial structures are properly preserved for accurate identification. Finally, advancements like microwave-assisted tissue processing and fixation are improving diagnostic turnaround times while maintaining tissue quality, representing significant progress in pathology practices.

Acknowledgement

None

Conflict of Interest

None

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