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Natural fixatives alternative to formalin in histopathology: A systematic review

Yee AWM, Oo PS, Aye SN, Lim WJ, Chee VCX, Krishnappa P

Med J Malaysia, 2023 Jan;78(1):98-108.
PMID: 36715199

INTRODUCTION: Since constant long-term exposure to formaldehyde endangers the health of laboratory personnel, sugar-based natural products have become interesting alternative fixatives to formaldehyde because of their preservative and antibacterial properties. However, there are controversial findings on the fixative effects of natural fixatives. This study systematically reviews the evidence comparing natural fixatives' types, dilutions, fixative properties and staining quality in normal tissues and histopathological specimens.
MATERIALS AND METHODS: A comprehensive search was performed for studies comparing the natural fixatives- and formaldehyde-fixed tissues using databases from inception to January 2022: PubMed, Ovid Medline and Google Scholar. Two independent reviewers did data extraction. The data were pooled for the type of natural fixatives, their concentrations and fixative qualities compared to formaldehyde.
RESULTS: Fifteen studies were included in this systematic review. Nine studies used one natural fixative with different dilutions, while six used several natural fixatives to compare their fixative properties with formaldehyde. The most used natural fixative was honey (n = 12) followed by jaggery (n = 8), sugar (n = 3) and others (n = 1). Honey showed the most promising results in fixation and staining, which are compatible with formalin. Jaggery and sugar also showed the possibility of replacing formaldehyde in tissue fixation and staining in smaller tissue samples.
CONCLUSION: Natural fixatives showed promising results in tissue fixation. However, optimising the concentrations and conditions of natural fixatives is difficult because of the different chemical constituents and production steps. More comprehensive studies are necessary for application.

Matched MeSH terms: Tissue Fixation/methods
A comparative study of microwave oven-assisted tissue processing and conventional method of tissue processing on turnaround laboratory time and morphological quality of tissue sections

FinNie O, Aye SA, Krishnappa P, Ravindran R

Med J Malaysia, 2023 Mar;78(2):202-206.
PMID: 36988531

INTRODUCTION: The purpose of tissue processing is to fix the tissue in a solid medium toenable thin sections. Conventional method of tissue processing is the standardized method of tissue processing which has been used for more than 10 decades. However, the conventional method is time-consuming, and the overall turnaround time for the histopathology report is at least two days. The objective of this study is to identify the protocol for tissue processing procedure using domestic microwave oven. To determine the tissue processing time when using domestic microwave oven. To compare the morphological quality of tissue slides made by domestic microwave oven and conventional method using automated tissue processor.
MATRIALS AND METHODS: The conventional protocol and three microwave protocols of tissue processing were used in this study. A pilot study was done prior to the real run to determine the baseline timing for microwave protocol. The baseline timing was fixed at 2 minutes,30 minutes,5 minutes and 25 minutes. The processing time of the microwave protocol was adjusted from 62 minutes to 70 minutes to 77 minutes by increasing the dehydration and wax impregnation time while the time for tissue fixation and clearing remain the same throughout all the microwave protocols.
RESULTS: The group 2 microwave protocol produced the sections that is closely comparable to group 1 conventional protocol. The morphological quality of histopathology slides is best observed when the processing time of microwave protocol is 62 minutes.
CONCLUSION: The most appropriate microwave protocol for tissue processing is group 2 as the morphological quality of histopathology slides are more superior than that of group 1 with an overall percentage of 80% of satisfactory slides in group 2 and 76.68% in group 1.

Matched MeSH terms: Tissue Fixation/methods
Immunostaining of formalin-fixed, paraffin-embedded tissues for malignant lymphomas

Lim YC, Phang KS, Cheong SK

Malays J Pathol, 1992 Dec;14(2):85-9.
PMID: 1304629

With the advent of new monoclonal antibodies that are applicable to formalin-fixed, paraffin embedded sections, immunophenotyping is becoming increasingly important in the diagnosis and classification of lymphomas. However, multiple factors such as fixation, trypsinization and even type of antibodies used have certain effects on the final outcome of the staining procedure. In this paper we report our experience and the problems encountered in our laboratory when we first tried to establish a workable immunostaining protocol for formalin-fixed, paraffin embedded tissue sections using the immunoalkaline phosphatase technique.

Matched MeSH terms: Tissue Fixation/methods
Fulltext In situ hybridisation: principles and applications

Looi LM, Cheah PL

Malays J Pathol, 1992 Dec;14(2):69-76.
PMID: 1304627

In situ hybridisation (ISH) is based on the complementary pairing of labelled DNA or RNA probes with normal or abnormal nucleic acid sequences in intact chromosomes, cells or tissue sections. Compared with other molecular biology techniques applicable to anatomical pathology, ISH enjoys better rapport with histopathologists because of its similarity to immunohistochemistry. It has the unique advantage over other molecular biology techniques--largely based on probe hybridisation with nucleic acid extracted from homogenised tissue samples--of allowing localisation and visualisation of target nucleic acid sequences within morphologically identifiable cells or cellular structures. Probes for ISH may bear radioactive or non-radioactive labels. Isotopic probes (3H, 32P, 35S, 125I) are generally more sensitive than non-isotopic ones but are less stable, require longer processing times and stringent disposal methods. Numerous non-isotopic labels have been used; of these biotin and digoxigenin are the reporters of choice. Optimised non-isotopic systems of equivalent sensitivity to those which use radioactive-labelled probes have been described. In ISH, finding the optimal balance between good morphological preservation of cells and strong hybridisation signals is crucial. Tissue fixation and retention of cytoskeletal structures, unfortunately, impede diffusion of probes into tissues. ISH sensitivity is also influenced by inherent properties of the probe and hybridisation conditions. Although ISH is largely a research tool, it is already making strong inroads into diagnostic histopathology. It has been applied for the detection of various infective agents particularly CMV, HPV, HIV, JC virus, B19 parvovirus, HSV-1, EBV, HBV, hepatitis delta virus, Chlamydia trachomatis, salmonella and mycoplasma in tissue sections.(ABSTRACT TRUNCATED AT 250 WORDS)

Matched MeSH terms: Tissue Fixation/methods*
Fulltext Molecular pathology quality control in Southeast Asia: Results of a multiregional quality assurance study from MASTER KEY Asia

Okuma HS, Yoshida H, Kobayashi Y, Arakaki M, Mizoguchi C, Inagaki L, et al.

Cancer Sci, 2023 Jun;114(6):2664-2673.
PMID: 36919757 DOI: 10.1111/cas.15790

Tissue specimen quality assurance is a major issue of precision medicine for rare cancers. However, the laboratory standards and quality of pathological specimens prepared in Asian hospitals remain unknown. To understand the methods in Southeast Asian oncology hospitals and to clarify how pre-analytics affect the quality of formalin-fixed paraffin-embedded (FFPE) specimens, a questionnaire surveying pre-analytical procedures (Part I) was administered, quality assessment of immunohistochemistry (IHC) staining and DNA/RNA extracted from the representative FFPE specimens from each hospital (Part II) was conducted, and the quality of DNA/RNA extracted from FFPE of rare-cancer patients for genomic sequencing (Part III) was examined. Quality measurements for DNA/RNA included ΔΔCt, DV200, and cDNA yield. Six major cancer hospitals from Malaysia, Philippines, and Vietnam participated. One hospital showed unacceptable quality for the DNA/RNA assessment, but improved by revising laboratory procedures. Only 57% (n = 73) of the 128 rare-cancer patients' specimens met both DNA and RNA quality criteria for next-generation sequencing. Median DV200 was 80.7% and 64.3% for qualified and failed RNA, respectively. Median ΔΔCt was 1.25 for qualified and 4.89 for failed DNA. Longer storage period was significantly associated with poor DNA (fail to qualify ratio = 1579:321 days, p

Matched MeSH terms: Tissue Fixation/methods
RCL2, a potential formalin substitute for tissue fixation in routine pathological specimens

Masir N, Ghoddoosi M, Mansor S, Abdul-Rahman F, Florence CS, Mohamed-Ismail NA, et al.

Histopathology, 2012 Apr;60(5):804-15.
PMID: 22320393 DOI: 10.1111/j.1365-2559.2011.04127.x

To investigate RCL2 as a fixative for tissue fixation in routine histopathological examination and to assess tissue suitability for ancillary investigations.

Matched MeSH terms: Tissue Fixation/methods*
Specimen shrinkage and its influence on margin assessment in breast cancer

Yeap BH, Muniandy S, Lee SK, Sabaratnam S, Singh M

Asian J Surg, 2007 Jul;30(3):183-7.
PMID: 17638637

The determination of tumour-free margin in breast cancer is crucial in deciding subsequent patient management. To exemplify the phenomenon of margin contraction during specimen preparation for histopathological analysis, we quantified the shrinkage of breast specimens as a result of formalin fixation.

Matched MeSH terms: Tissue Fixation/methods*
Microwave-stimulated formaldehyde fixation of experimental renal biopsy tissues: computerised morphometric analysis of distortion artefacts

Looi LM, Loh KC

Malays J Pathol, 2005 Jun;27(1):23-7.
PMID: 16676689

Although microwave irradiation has been used in the histopathology laboratory for several years, there has been minimal published experimental data on its effects on the technical and staining quality of histological sections. Furthermore, it has not been clear whether the advantages gained in reduction of fixation and staining duration has been at the expense of increasing architectural distortion to the tissues. We report here our experience with computerised morphometric analysis to investigate glomerular artifacts caused by microwave-stimulated fixation of renal tissues. 39 rat and 33 human autopsy kidney samples were subjected to (1) fixation in neutral buffered formaldehyde (control), (2) microwave-stimulated fixation followed by neutral buffered formaldehyde, and (3) neutral buffered formaldehyde followed by microwave irradiation. In addition, the effect of post-fixation in 70% ethanol was also investigated. Microwave irradiation was delivered through a dedicated laboratory microwave oven at 80% power and at 55 degrees C for 3 minutes. The different fixation methods were compared with regards to shrinkage (distortion) to glomerular structures (glomeruli and Bowman's spaces) on H and E sections, as determined by morphometric image analysis using a temporary assembled-system consisting of a trinocular microscope, a digital video camera and personal computer. A FlashPoint VGA 3.3 film-grabber card was used to capture images for morphometric analysis by using a Scion Image program. Morphometric analysis of glomerular structures showed that microwaves caused more shrinkage to the area bounded by the Bowman's capsule than the glomerulus proper, but post-fixation with ethanol reduced this shrinkage. These findings have implications on the logistics of tissue preparation of renal biopsies in clinical practice.

Matched MeSH terms: Tissue Fixation/methods*
Fulltext Structural Studies of Epithelial Mesenchymal Transition Breast Tissues

Mohd Sobri SN, Abdul Sani SF, Sabtu SN, Looi LM, Chiew SF, Pathmanathan D, et al.

Sci Rep, 2020 02 06;10(1):1997.
PMID: 32029810 DOI: 10.1038/s41598-020-58932-5

At the supramolecular level, the proliferation of invasive ductal carcinoma through breast tissue is beyond the range of standard histopathology identification. Using synchrotron small angle x-ray scattering (SAXS) techniques, determining nanometer scale structural changes in breast tissue has been demonstrated to allow discrimination between different tissue types. From a total of 22 patients undergoing symptomatic investigations, different category breast tissue samples were obtained in use of surgically removed tissue, including non-lesional, benign and malignant tumour. Structural components of the tissues were examined at momentum transfer values between q = 0.2 nm-1 and 1.5 nm-1. From the SAXS patterns, axial d-spacing and diffuse scattering intensity were observed to provide the greatest discrimination between the various tissue types, specifically in regard to the epithelial mesenchymal transition (EMT) structural component in malignant tissue. In non-lesional tissue the axial period of collagen is within the range 63.6-63.7 nm (formalin fixed paraffin embedded (FFPE) dewaxed) and 63.4 (formalin fixed), being 0.9 nm smaller than in EMT cancer-invaded regions. The overall intensity of scattering from cancerous regions is a degree of magnitude greater in cancer-invaded regions. Present work has found that the d-spacing of the EMT positive breast cancer tissue (FFPE (dewaxed)) is within the range 64.5-64.7 nm corresponding to the 9th and 10th order peaks. Of particular note in regard to formalin fixation of samples is that no alteration is observed to occur in the relative differences in collagen d-spacing between non-lesional and malignant tissues. This is a matter of great importance given that preserved-sample and also retrospective study of samples is greatly facilitated by formalin fixation. Present results indicate that as aids in tissue diagnosis SAXS is capable of distinguishing areas of invasion by disease as well as delivering further information at the supramolecular level.

Matched MeSH terms: Tissue Fixation/methods