Authors (including presenting author) :
Wong CK(1), Lam Y(1), Kwan YKA(2)
Affiliation :
(1)School of Science and Technology, Hong Kong Metropolitan University, (2)ALIS PHARMA
Introduction :
In the histopathology laboratory, there are always challenges and limitations of adipose tissue processing, no matter in standard traditional tissue processes or rapid tissue processing machines. On account of the fat component being less amenable to fixation than other tissue components, formalin which is a common fixative, may not penetrate adipose tissue effectively and leading to incomplete fixation and potential tissue artifacts. Incomplete fixation of adipose tissue would lead to artifacts creation, these artifacts can affect the quality of histological slides for microscopic examination.
Adipose tissue might feature difficulty in sectioning and embedding, due to its soft and malleable nature. Moreover, fatty tissue surrounds lymph nodes in the body, and various diseases can affect the lymphatic system, for example, lymphadenopathy, lymphedema, and lymphomas, among others, assessing any abnormalities in the adipose tissue can indicate various diseases of the lymphatic system. An in-house fatty tissue processing method is developed in this research project. Pig is chosen for the animal model for the project. Pigs are a desirable alternative for simulating human diseases since they resemble people more physically, physiologically, and pathophysiologic than small rodents like mice. They are threefold closer to humans on the nucleotide level than are mice.
In this study, a rotational tissue processor that also features a unique characteristic is applied. It has a canted chamber to enhance reagent distribution as well as reduce tissue processing time, and eventually can allow for high-quality processing results. In contrast to the traditional method, which may not be optimal for adipose tissue, this automatic processor is beneficial to specific tissue types such as brain tissue or adipose tissue. These benefits have been addressed in the context of this research project. Validation of a brand-new automatic tissue processor is crucial, because each parameter of the equipment setting can affect the diagnostic results, and the older or other tissue processor’s parameters and settings are not suitable for application.
Objectives :
There are two major objectives in this project, include developing an in-house fatty tissue processing method with a pig model; and to validate an automatic tissue processor. The scope includes utilizing the automatic tissue processor to streamline the processing of adipose tissues, aiming to improve turnaround time (TAT) and reduce reagent usage without compromising the quality of histological sections. This research project also encompasses assessing the performance and reliability of the an automatic tissue processor in handling adipose tissues by implementing rigorous quality control measures to ensure consistency and accuracy in tissue processing and histological evaluations.
This research project delineates comprehensive and optimal procedures, offering a reliable reference for handling adipose tissue to attain superior clinical outcomes. Succeeds with the validation of the tissue processor ensures that the automatic tissue processor meets the expected performance. Complete and document a validation report of this process which serves as a crucial record for regulatory compliance and ongoing quality control.
Methodology :
Utilizing pig tissue models, chosen for their anatomical and physiological similarities to humans, the study employed adipose tissue to develop a comprehensive in-house processing protocol. 50 pig tissue samples were processed using an automatic processor, followed by critical embedding, sectioning, and hematoxylin and eosin (H&E) staining, among others. The research employed a quantitative approach, using a scale score and independent t-tests to assess the impact of varied parameters on tissue preservation and quality. Rating scales for paraffin sectioning and H&E staining results contributed to quantitative analysis, enhancing the reliability and accuracy of outcomes.
Result & Outcome :
Adipose tissue is difficult to be processed in the field of histology, due to the inherent difficulty in penetrating the fat, this can lead to under-fixation or poor tissue processing. Moreover, adipose tissues pose high lipid content that will cause paraffin microtome sectioning difficulty, which can interfere with immunohistochemistry or immunofluorescence studies. These challenges are encountered in both traditional manual processes and rapid automation tissue processing.
To address these issues, this study successfully developed an in-house fatty tissue processing method. The method encompassed optimal settings and handling techniques for adipose tissue, ensuring the acquisition of high-quality sample results.
Key aspects have been considered include the choices and time parameters of fixation, dehydration, clearing, and infiltration, among others. A specific protocol was developed which demonstrated a successful paraffin microtome sectioning process with minimal artifacts and high consistency. A good experimental results indicated reliable and reproducible microtome sectioning results, further validating the efficacy of the developed protocol in achieving high-quality FFPE sections.
Under microscopic examination, the processing method developed had successfully displayed the adipose tissue structure of individual adipocytes, with the nuclei of the adipocytes visible and stained blue by hematoxylin. The overall quality of the H&E staining appeared good, with a clear background and excellent visualization of the fatty tissue morphology. A validation report for the automatic tissue processor was completed which served as a crucial record for regulatory compliance and ongoing quality control.
As a conclusion, an in-house fatty tissue processing method was developed using the pig model with a validated automatic tissue processor. Findings of studies have generated suggestions to address difficulties in laboratory technology for providing quality pathology service and better patient management.
