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Evaluating the Efficacy of a Formalin Alternative in Fixing Pathological Tissues for Histological and Molecular Diagnoses
Biomed Sci Letters 2023;29:48-52
Published online March 31, 2023;  https://doi.org/10.15616/BSL.2023.29.1.48
© 2023 The Korean Society For Biomedical Laboratory Sciences.

Min-A Je1,2,§,*, Haneul Lee1,§,**, Heechul Park1,2,*, Dong Hyeok Kim1,2,*, Yeongdon Ju1,2,*, Jaewon Lim1,***, Sunghyun Kim1,***and Jungho Kim1,†,***

1Department of Clinical Laboratory Science, College of Health Sciences, Catholic University of Pusan, Busan 46252, Korea
2Clinical Trial Specialist Program for In Vitro Diagnostics, Brain Busan 21 Plus Program, Graduate School, Catholic University of Pusan, Busan 46252, Korea
Correspondence to: Jungho Kim. Department of Clinical Laboratory Science, College of Health Sciences, Catholic University of Pusan, Busan 46252, Korea.
Tel: +82-51-510-0660, Fax: +82-51-510-0568, e-mail: jutosa70@cup.ac.kr
*Graduate student, **Ungraduate student, ***Professor.
§These authors have contributed equally to this work.
Received February 24, 2023; Revised March 29, 2023; Accepted March 29, 2023.
This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/3.0/) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.
 Abstract
Formaldehyde use is associated with serious health risks, which can affect medical personnel and technicians. Therefore, we investigated the efficacy of an alternative fixative, with respect to two types of formalin fixatives, by hematoxylin and eosin (H&E) staining, periodic acid Schiff (PAS) staining, immunohistochemical (IHC) staining, and RNA extraction. For H&E staining, the circular nucleus was stained dark blue by the basic dye hematoxylin and the cytoplasm was stained red by the acid dye eosin in all three fixative samples. No difference was found in the Duksan General Science (DGS), Sigma-Aldrich, and Core-Fix fixative samples (Corebiotech) used to fix kidney tissue, after PAS staining. IHC staining showed that CD4 was significantly increased in the lippolysaccharide (LPS)-treated group compared to the control group (vehicle), confirming the changes in specific molecules. The quantity and quality of RNA from tissues fixed in the three types of fixatives were evaluated. The average concentration of RNA was 106 ng/μL and average purity at A 260/280 ratio was 1.7~2.0, regardless of fixative used. For quality of protein, glyceraldehyde 3-phosphate dehydrogenase (GAPDH) protein was confirmed by Western blotting. In conclusion, Core-Fix can be used as a fixative for pathological tissues, in histological and molecular diagnoses.
Keywords : Formaldehyde, Formalin-fixed paraffin embedded (FFPE) tissues, Histopathology, RNA extraction

Histological analysis is a basic research method for examining various tissues that make up living organisms. Fixation, a process that prevents tissue degeneration, is the basis of histological studies for the accurate morphological identification of tissues and cells (Kothmaier et al., 2011; Qin et al., 2021). Formalin fixation and paraffin embedding are the most commonly used histological methods of tissue preservation (Bass et al., 2014). Tissue fixation using formalin effectively suppresses tissue degeneration and preserves cell structure (Bronsert et al., 2014). Tissue fixation is one of the most important aspects of a reliable histological sample preparation process and is an important factor in providing accurate results in research and diagnosis (Puchtler and Meloan, 1985; Zeng et al., 2013). Incomplete fixation may make it impossible to accurately identify the tissue, because morphological preservation of the tissue is not achieved. Ideally, the structure, shape, and position of the tissue and cells should remain intact, and the loss of both intracellular and extracellular characteristic molecules should be prevented after fixation. In addition, rational fixation maintains tissue antigenicity, or minimizes antigenic denaturation, and prevents diffusion or rearrangement of substances in tissues (Qin et al., 2021). Depending on the characteristics of the fixative, differences may be observed in the tissue, for example, loss of specific molecular substances in the fixed tissue, expansion or contraction of tissue during tissue processing, differences in histological or immunohistochemical (IHC) staining, accuracy of the biochemical reaction process, preservation of organelle structure, etc (Puchtler and Meloan, 1985; Becker et al., 2007; Nirmalan et al., 2009; Ludyga et al., 2012).

Formaldehyde is a representative chemical substance that can affect the health of medical personnel and technicians (Alexandersson and Hedenstierna, 1989). Exposure to formaldehyde can cause severe irritation to the eyes, nose, and bronchi, causing watery eyes, dizziness, and headache. Repeated exposure to formaldehyde can cause dermatitis and asthma. Formaldehyde may also cause cancer, and the incidence of nasopharyngeal cancer is significantly high in workers who use formaldehyde (Hauptmann et al., 2004). The International Agency for Research on Cancer (IARC) and the Ministry of Employment and Labor in Korea have classified formaldehyde as a carcinogen (Cancer, 2013; Hygienists, 2013). The US government's Industrial Hygiene Experts' Association classifies it as a presumptive carcinogen A2 (Hygienists, 2013). Therefore, in this study, the effectiveness of Core-fix was evaluated, compared with conventional formaldehydes, as a tissue fixative for pathological diagnosis (Table 1).

Characteristics of the formalin fixatives and the alternative fixative

Fixative Composition Vendor
Formaldehyde Solution 10% = (EP) Water
Ethyl alcohol
Glyoxal
10% formaldehyde
Methanol solution
Duksan
General
Science (DGS)
Formaldehyde solution Water
Ethyl alcohol
Glyoxal
37% formaldehyde
10~15% methanol
Sigma-Aldrich
Core-Fix Water
Glyoxal
< 1% formaldehyde
Corebiotech


After excising the liver, kidney, spleen and stomach of the experimental mouse, the organs were divided into three groups (0.5횞0.3 cm each) and fixed in one of the three: formaldehyde solution from Duksan General Science (DGS, Seoul, Korea), formaldehyde solution from Sigma-Aldrich (Sigma-Aldrich, Saint Louis, MO, United States), or Core-fix (Corebiotech, Gwangju, Korea), for 8 h at room temperature. This study was approved by the Animal Experimental Ethics Committee of the Catholic University of Pusan (CUP AEC 2022-002).

The fixed sample underwent dehydration to completely remove moisture by moving from a low-concentration alcohol to a high-concentration alcohol. After the tissue was placed in xylene solution, the transparent agent contained in the tissue was replaced with a penetrant to undergo paraffin infiltration to harden the tissue. The process from fixation to paraffin infiltration was carried out for 14 h using Peloris Quick Tips (Leica, Germany), and the tissue embedding device Histocentre 3 (Thermo Fischer Scientific, USA) was used for paraffin embedding.

To evaluate stainability, hematoxylin and eosin (H&E) staining and periodic acid-Schiff (PAS) staining were performed on the sliced tissue slides. Liver, kidney, spleen and stomach tissues from the three groups (DGS, Sigma-Aldrich, or Core-Fix) were subjected to H&E staining and confirmed with an optical microscope at 400X. The structure of the hepatic lobule and portal area were well maintained in all three groups (Fig. 1 A-C). The glomerular structure of the kidney tissues was well preserved in all three groups (Fig. 1 D-F). The spleen and stomach tissues in all three groups showed the same staining properties (Fig. 1 G-L). H&E staining showed that the circular nuclei were stained dark blue by the basic dye hematoxylin, and the cytoplasm were stained red by the acidic dye eosin; no differences in staining were observed among the three groups. Further, kidney tissues in the three groups were stained with PAS and examined under an optical microscope at 400X. The basement membrane was stained red with Schiff's reagent and the nuclei were stained dark blue with Harris hematoxylin. These results confirmed that there was no difference in staining based on the type of fixative (Fig. 2).

Fig. 1. Hematoxylin and Eosin staining. Formalin-fixed Paraffin-embedded (FFPE) tissues fixed in formaldehyde solutions from (A, D, G, J) Duksan General Science (DGS) and (B, E, H, K) Sigma-Aldrich, and for paraffin-embedded tissues fixed in (C, F, I, L) Core-Fix. Liver (A, B, C), kidney (D, E, F), spleen (G, H, I), and stomach (J, K, L) tissues of mice were observed under an optical microscope (400X).

Fig. 2. Periodic acid-Schiff staining. Formalin-fixed Paraffin-embedded (FFPE) tissues fixed in formaldehyde solutions from (A) Duksan General Science (DGS) and (B) Sigma-Aldrich, and for paraffin-embedded tissues fixed in (C) Core-Fix. Kidney tissue of mice was observed under an optical microscope (400X).

To confirm the immuno-histological differences based on the type of fixative used, immunohistochemical (IHC) staining for CD4 was performed. IHC staining for CD4 after using two conventional fixatives (DGS and Sigma-Aldrich) and a fixative substitute (Core-Fix) in the kidney confirmed that CD4 (brown color) was significantly increased in the lipopolysaccharide (LPS)-treated group compared to the control group (vehicle) (Fig. 3).

Fig. 3. Immunohistochemical staining. Formalin-fixed Paraffin-embedded (FFPE) tissues fixed in formaldehyde solutions from (A, D) Duksan General Science (DGS) and (B, E) Sigma-Aldrich, and for paraffin-embedded tissues fixed in (C, F) Core-Fix. Kidney tissue of mice was observed under an optical microscope (400X). (G) Graph for antibody measurements of CD4. *P < 0.05, **P < 0.01, ***P < 0.001.

Next, the purity and concentration of the extracted RNA were compared and analyzed to understand the effect of the tissue fixative used in the experiment on the molecular pathological test. The purity and concentration of the RNA were determined by measuring the absorbance values at 260 and 280 nm. Results for RNA extraction using three fixatives, DGS, Sigma-Aldrich, and Core-Fix, showed that the average A260/280 nm ratio of the samples was 1.7~2.0 in each fixative. This indicates that the samples were not contaminated with protein (Table 2). The quality of the extracted protein and the results of GAPDH protein were investigated using Western blotting. The band of GAPDH protein in all three groups showed the same properties (Fig. 4).

RNA concentrations and purities in the formalin fixatives and the alternative fixative

Fixative Organ RNA concentration (μL) A260/280 ratio
Formaldehyde solution 10% = (EP)
(Duksan General Science)
Kidney 27.9 1.8
Spleen 150.5 1.9
Liver 159.8 1.8
Mean ± SD 112.7±73.6 1.8±0.05
Formaldehyde solution (Sigma-Aldrich) Kidney 27.9 1.7
Spleen 120.8 1.9
Liver 144.3 2.0
Mean ± SD 97. 7±61.5 1.8±0.15
Core-Fix (Corebiotech) Kidney 39.5 1.7
Spleen 125.5 2.0
Liver 138.6 2.0
Mean ± SD 101.2±53.8 1.9±0.17


Fig. 4. GAPDH protein examined by Western blotting using Formalin-fixed Paraffin-embedded (FFPE) tissues fixed in formaldehyde solutions from Duksan General Science (DGS) and Sigma-Aldrich, and for paraffin-embedded tissues fixed in Core-Fix.

Among the harmful factors to which hospital workers are exposed, formaldehyde is a representative chemical factor (Buesa, 2008). Since formaldehyde is widely used for sterilization, storage, and inspection of tissues, it poses as a threat to the health of workers handing formaldehyde (Costa et al., 2008). Although there have been studies to improve the working environment by measuring the concentration of formaldehyde being exposed, studies comparing and analyzing the effectiveness of alternative agents are scarce.

In this study, paraffin blocks fixed with three different fixatives (DGS, Sigma-Aldrich, and Core-Fix) were sectioned into 4-關m slices to produce slides with general staining (H&E) and special staining (PAS). No differences were observed in the staining based on the fixative used. All the three fixatives had similar RNA concentrations. The purity showed excellent results of 1.7 to 2.0 at A260/280 nm, regardless of the fixative used. Various special staining such as acid fast stain, masson trichrome stain and toluidine blue stain should be further investigated with Core-Fix.

In conclusion, no special difference between existing fixatives and Core-Fix (formalin-free fixative) could be found in specimens and biopsies. Thus, for biopsies performed in outpatient departments, endoscopy rooms, and ultrasound rooms, even Core-Fix can be used instead of the existing formaldehyde fixatives.

ACKNOWLEDGEMENT

None.

CONFLICT OF INTEREST

The researcher claims no conflicts of interest.

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