Fixation of biopsy tissues is a crucial step before tissue processing for histopathological examinations. Tissue fixation helps to preserve the tissue in a life-like state, prevent autolysis and putrefaction, alter the refractive index, and provide mechanical rigidity to maintain its integrity during consecutive stages of processing. ¹ Ideally, fixation should maintain clear and consistent morphological features of the tissue with minimal disruption to its cellular-extra cellular relationships and chemical composition after staining for microscopic examination. ²

Since the late 19 ^thcentury, formalin has been the fixative of choice following the accidental discovery by Ferdinand Blum, that a diluted solution of formaldehyde not only “fixed” tissues but also provided excellent staining with hematoxylin and aniline dyes. ³ Formaldehyde acts by the formation of methylene bridges between the amino acids of the protein molecules present in the tissues, thereby forming crosslinks. ² The routine fixative (neutral-buffered formalin of pH 7.2–7.4), which is a 10% solution of formalin (approximately 4% formaldehyde solution), is readily available, easy to use, cost-effective, and provides adequate fixation for good quality histology. However, it was classified as a carcinogen by the U.S Environmental Protection Agency when there was prolonged or high exposure, with a permissible level of 0.75 ppm average daily exposure. ⁴ Formaldehyde was classified as a Class 1 human carcinogen with the potential to cause different neoplasms including nasopharyngeal carcinoma by the International Agency for Research on Cancer (IARC). ⁵ Hence, the search for safe alternative fixatives has gained interest as pathologists and laboratory technicians are at a high risk of toxicity from constant exposure to formalin. Recent literature shows many studies exploring the possibility of using natural eco-friendly substances such as honey and jaggery to fix tissues. ⁶ , ⁷ , ⁸

Honey is an organic, naturally sweet substance produced by honeybees by the collection of plant nectar which consists of a mixture of sugars and other compounds such as lysozymes, vitamins, minerals, trace elements, acids, hydrogen peroxide, etc. ⁹ Literature shows that honey exhibits antibacterial properties due to high acidity and hygroscopic nature and also anti-autolytic and tissue hardening properties thus satisfying the requirements to be a fixative. ⁹ The staining properties and tissue morphology of histological sections of tissues fixed in honey have been found to be at par with formalin. ⁸ , ¹⁰

Many studies are continuously being done to prove the efficiency of honey as a good fixative for cytology and histology. However, the standardization has not been done so far. The present study is aimed to standardize honey as a tissue fixative for histopathology. Cell shrinkage, an inevitable effect of fixation, is an important characteristic when choosing a fixative so that the histomorphometric patterns can be reproduced.

In vitro study was conducted in the Department of Oral Pathology and Microbiology at a Private Dental College in Chennai, India.

Sample size calculation

The sample size per group was estimated as 29 after setting α error as 5% and the power of the study as 90%.

Tissue preparation

The samples for the in vitro study were oral tissues of goat (buccal mucosa), which were remnants from the meat sold commercially for the human consumption at the butcher's shop. The tissues were collected at the time of slaughter of the animal, which was done for the purpose of sourcing meat for the consumption. The fresh samples of 1 cm × 1 cm × 1 cm dimension were fixed straightaway by immersing them in containers with 10% honey and 10% formalin solution (Oxford laboratory, Thane, India), respectively. Ten percentage honey was prepared by mixing 10 mL honey with 90 mL of distilled water. Store-bought processed honey (Dabur honey, Dabur India Limited, Solan, India) was used in this study as unfiltered honey may contain various artifacts and viable spores like clostridia causing false-positive reactions. ⁹ The tissues were fixed in the respective solutions for 24 h at the room temperature and were subjected to routine processing. The adequately fixed tissues were kept under running water for 10 min to remove the fixative from the tissues. Tissue dehydration was done subsequently by immersing the tissues in ascending concentration grades (70%, 90%, and 100%) of isopropanol (EMPLURA, Merck Life Science Private Limited, Mumbai, India) for 30 min each. Removal of alcohol from the tissues was done by immersing them in the clearing agent xylene (Merck Life Science Private Limited, Mumbai, India) for 1–2 h. Molten paraffin wax (Merck Life Science Private Limited, Mumbai, India) in a wax bath (54–55°C melting point) was used to impregnate the tissues by keeping them in it for a few hours to remove xylene. The wax-infiltrated tissues were then embedded in paraffin wax with the help of L-shaped metal mounts. 4 μm sections were cut from the tissue blocks with a rotary microtome (Leica RM 2155, Ambala Cantt, India). The tissue sections were mounted on glass slides for viewing in a light microscope. Routine staining with hematoxylin (Nice Chemicals Pvt. Ltd, Kochi, India) and eosin (Thermo Fisher Scientific India Pvt Ltd, Mumbai, India) was done Figure 1and Figure 2. Figure 1

H and E stained histological section of tissue as viewed in x10 light microscope – (a) tissue fixed in formalin and (b) tissue fixed in honey.

The descriptive statistics, i.e., mean, standard deviation, median (P50), and IQR = P25–P75 computed for the different morphometric variables (CA, CP, NA, NP, Cyt A, N/C) for both the honey-fixed and formalin-fixed groups are represented in Table 2and Table 3.{Table 2}{Table 3}

The intergroup comparison for the different morphometric variables was performed using Mann–Whitney U-test which showed that there was a statistically high significant difference between the groups for the variables: CA (P < 0.0001), CP (P < 0.0001), NP (P = 0.0006), Cyt A (P < 0.0001) and N/C ratio (P < 0.0001).

The probability for the honey-fixed group to have higher values than the formalin-fixed group as elucidated using the Mann–Whitney U-test was found to be 0.515 for NA, 0.542 for NP and 0.684 for N/C ratio.

The probability for the formalin-fixed group to have higher values than the honey-fixed group as elucidated using the Mann–Whitney U-test was found to be 0.777 for CA, 0.716 for CP and 0.782 for Cyt A.

The data distribution pertaining to the different morphometric variables (CA, CP, NA, NP, Cyt A, and N/C) for the honey-fixed and the formalin-fixed groups are depicted using box and whisker plot Figure 4. Figure 4

Box and Whisker plot for data distribution of, (a) Cell area and cell perimeter for honey and formalin fixatives, (b) Cytoplasmic area and N/C ratio for honey and formalin fixatives, (c) nuclear area and nuclear perimeter for honey and formalin fixatives.

The growing concern regarding the biohazards associated with regular formalin usage in the laboratories has fueled the search for alternate fixatives that can effectively replace formalin. Literature shows that exogenous formaldehyde exposure may result in irritation in the eyes and upper respiratory tract. The IARC, based on comprehensive results from extensive human and animal studies, concluded that formaldehyde caused nasopharyngeal cancer, leukemia and was positively associated with sinonasal cancer. ¹¹ It is cytotoxic, genotoxic and can cause mutagenic changes and DNA damage. ¹² Earlier studies have shown that the formation of monoadducts, DNA-DNA crosslinks, DNA-protein crosslinks while binding to DNA has been related to the genotoxic effects of formaldehyde. ¹³ Formalin being a cross-linking fixative binds to amino acids and nucleotides by the formation of methylene bridges and blocks the retrieval of nucleic acids and also degrades the material making them unsuitable for further molecular studies. Similarly, it has deleterious effects on the resolution of proteins in the tissues making it difficult for immunohistochemistry. ¹⁴

Honey, an organic product, has been used for centuries for mummification of human cadavers and also to preserve meat because of its high acidity, antibacterial, and hygroscopic properties. ¹⁰ In addition, honey also exhibits anti-autolysis and tissue-hardening properties. ⁹ Taking these properties into consideration, honey is being used in various studies to see if it is an effective alternative to formalin as a tissue fixative. A pilot study was conducted earlier in the department to compare the fixative properties of honey and formalin qualitatively. ¹⁵ The present study was conducted as an attempt to standardize honey as a tissue fixative using the morphometric analysis.

In the present study, the tissues were adequately fixed in formalin as well as processed honey. Processed honey was preferred over the unprocessed one as unprocessed honey has been found to show increased fixation artifacts in tissues as compared to processed honey. Furthermore, greater slit like spaces were also evident in the basement membrane due to epithelial shrinkage with unprocessed honey. ⁹

Morphometric analysis done in the present study enabled in the identification of dimensional changes in the epithelial cells when fixed with honey. The probability of epithelial cells fixed with formalin to have greater CA, CP, and Cyt A was 0.777 (77.1%), 0.716 (71.6%), and 0.782 (78.2%), respectively, when compared to those tissues fixed with honey. This was suggestive of shrinkage corresponding to cytoplasm in the tissues fixed in honey. Similarly, the probability of the epithelial cells tending to have greater NA, NP and N/C ratio was 0.515 (51.5%), 0.542 (54.2%), and 0.682 (68.2%), respectively, for tissues fixed in honey. Since the morphometric method was not used for comparison in the pilot study done earlier, only qualitative analysis was possible, and no change was observed visually in the epithelium. ¹⁵ The result suggests that honey is a better nuclear fixative than formalin which is in accordance with the pilot study that showed honey is a good nuclear fixative. ¹⁵ The acidic component of honey provides for its low pH and usually fixatives with low pH act as good nuclear fixatives as they do not favor preservation of cytoplasmic constituents. ⁹ Hence, the probability of shrinkage of epithelium during the fixation of tissues in honey should always be taken into account.

The concentration of honey should also be considered while using it as a tissue fixative. The results of the previous studies were based only on qualitative comparison between honey and formalin fixed tissues. Literature has shown that honey at different concentrations modified the various aspects of fixation. At higher concentrations (70%–100%), honey was suitable for long-term gross preservation with moderate tissue morphology and at lower concentrations (20%–50%), it exhibited excellent staining characteristics. ¹⁶ When 10% honey was used as a fixative, both the stain uptake and preservation of tissue architecture were at par with tissues fixed in formalin though collagen fibers in the connective tissue gave a hyalinized appearance. ¹⁴ , ¹⁷

A study by Patil et al. showed that though tissues fixed in 20% honey over 6-month duration relatively retained their gross morphology, there was a gradual reduction in the cellular and nuclear clarity. Furthermore, evident cellular and nuclear shrinkage was observed in honey-fixed tissues in comparison to formalin on microscopic examination. ¹⁸ In the present study, some amount of shrinkage was evident in the cytoplasm of epithelial cells of honey-fixed tissues (24 h) on morphometric analysis. However, the extent of shrinkage in honey over the long periods of fixation is yet to be analyzed. The dimensional changes in the connective tissue were not analyzed in this study due to homogenization observed in honey fixed tissues. However, it is safe to state that honey would effectively satisfy all the properties of a fixative if there were methods to eliminate homogenization.

Although formalin fixation has some undermining effects on the extraction of nucleic acids and antigen retrieval for immunohistochemical staining, it is still being used. ¹⁴ Earlier studies have shown that tissues fixed in honey are suitable for IHC as both cytoplasmic and nuclear antigen expressions were effectively demonstrated. ¹⁵ , ¹⁹ , ²⁰ Similarly, although honey has shown effective nuclear fixation, retrieval of nuclear material for downstream applications and molecular studies from tissues fixed in honey requires further analysis.

This study has shown that honey is a better nuclear fixative than formalin. The probability of epithelial cells to have greater NA, NP, and N/C ratio is about 50%–60% in honey-fixed tissues. While, the probability of epithelial cells in formalin-fixed tissues tending to have greater CA, CP, and Cyt A is about 70%. Hence, researchers should be mindful of the cytoplasmic shrinkage of epithelial cells in honey-fixed tissues when compared to formalin fixation. By including tissues of other animal species and human tissues with varying concentrations of honey, the study may be expanded in future. This would provide further standardization of honey as a fixative.

Financial support and sponsorship

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Conflicts of interest

The authors of this manuscript declare that they have no conflicts of interest, real or perceived, financial or non-financial in this article.