Efficacy of Kumkum as a Surrogate for Eosin in Routine Histological Sections: An Observational Study

1. Assistant Professor, Department of Pathology, Chettinad Academy of Research and Education, Chettinad Hospital and Research Institute, Chengelpattu, Tamil Nadu, India. 2. Assistant Professor, Department of Pathology, Chettinad Academy of Research and Education, Chettinad Hospital and Research Institute, Chengelpattu, Tamil Nadu, India. 3. Senior Resident, Department of Pathology, Chettinad Academy of Research and Education, Chettinad Hospital and Research Institute, Chengelpattu, Tamil Nadu, India. 4. Allied Health Science Student, Department of Pathology, Chettinad Academy of Research and Education, Chettinad Hospital and Research Institute, Chengelpattu, Tamil Nadu, India.

Abstract

Introduction: Stains are a crucial component of laboratory procedure. They help in highlighting the different tissues, both normal and abnormal, which plays an important role in diagnostic histopathology. Synthetic dyes are utilised for majority of the stains in histology. Among the natural surrogates studied, Kumkum is less researched.

Aim: To compare the staining characteristics of Kumkum, which imparts red colour as an alternative to eosin.

Materials and Methods: This observational study was conducted at Histopathology section, Pathology department at Chettinad Hospital and Research Institute for a period of six months from January 2022 to June 2022. In 50 tissue blocks, each was made into two sections and one was stained with routine Haematoxylin and Eosin (H&E), while the other was stained with Haematoxylin and Kumkum solution (H&K). The Cellular Architecture (CA) (based on distinct or indistinct nucleus, cytoplasm) and quality of staining (poor, satisfactory or good) of H&E and H&K solution were compared and a scoring system was given. The scores were analysed with Chi-square test using SPSS software (version 25).

Results: Out of 100 slides, 45 (90%) stained with H&E and 48 (96%) with H&K showed distinct CA. A total of 47 slides (94%) stained with H&K and 41 (82%) with H&E showed good quality of staining.

Conclusion: Kumkum solution appears to be an efficient counterstain in place of eosin in highlighting the normal structures in histopathological sections. It highlighted the RBCs in the arteries and cytoplasm of the cells in glandular and squamous epithelium better than or equally good as eosin.

Counterstain, Maceration, Staining, Utility of Kumkum

Histology or histopathology is the microscopic examination of tissues and cells, even plants and animals. It is done by process of fixing, sectioning, staining and viewing under a microscope. Diagnostic histopathology is an important step in the management of the patient (1). Staining is useful for microscopy technique and employed to improve contrast in the material, often at the microscopic level. Stains and dyes are often used in biological tissues to highlight structure. Stains can be classified as acidic, basic, or neutral. The colour which is produced depends on the chemical nature of the stains and the cellular constituents. The staining method facilitates diagnosis by increasing the visibility of cells under a microscope. Biological dyes are classified into two categories; natural and artificial. Natural dyes are taken from natural resource; the most dominant natural dye in the histopathology laboratory is haematoxylin. Artificial dyes are derived from chemical agents (2).

H&E stain is the amalgamation of two histological stains. Haematoxylin, a basic dye is from natural dyes obtained from barks of Haematoxylon campechianum and also extract from the logwood tree, stains nucleus giving it a bluish-purple colour (3). Eosin is one of the acidic florescent chemical compounds that binds by basic or eosinophilic compounds and stains cytoplasm, pink to orange and nuclei in dark purple colour. It is one of the commonest counterstain used in routine histopathological sections (3).

Recently, researchers are debating that use of natural substances like beetroot, saffron, rose and turmeric in staining tissues [4,5]. In natural substances, Kumkum is one of the compounds that can be surrogative for eosin as it imparts red to dark pink colour to the tissues. The preparation of Kumkum powder can be by both commercial method as well as natural method. Mainly, Kumkum is taken from saffron flower and pinch of turmeric is added in the commercially available sachets. Kumkum is an eco-friendly compound, low cost, readily available, non toxic and organic stain (6).

To our knowledge, only few studies are available exploring Kumkum solution utilities in staining the different tissues in histopathology. Lavanya A et al., concluded the utility of dual staining property of kumkum for the differentiation of the components of tooth, bone and soft tissue structures in histostaining of oral tissues which facilitates the diagnosis of fibro-osseous lesions, bony, collagen and muscular pathologies (6). Navya N et al., concluded that Kumkum appears to be an efficient counterstain for demonstrating various structures in histopathology sections of cervix tissue (7). The current study is an attempt to compare the staining characteristics of eosin and Kumkum and prove that Kumkum is the best alternative to eosin. The present study highlights the novel use of Kumkum solution in the field of histopathology for the purpose of staining.

Material and Methods

The observational study was conducted at Histopathology section- Pathology Department, Chettinad Hospital and Research Institute, from January 2022 to June 2022. The institutional human ethics committee (IHEC-I/0573/22) reviewed the proposal and approved this study. A total of 50 tissue sections were analysed and used for staining which included normal cervix, endometrium, ovaries, artery, smooth muscle and nerve bundles. From each tissue block two sections were taken in which one was stained with routine H&E and other with H&K (Kumkum) stain. A total of 100 slides were used in two groups and the CA and staining efficacy was compared by two pathologists independently and blinded.

Inclusion criteria: Normal and well-fixed cervix, endometrium, ovaries, artery, smooth muscle, skeletal muscle and nerve bundles.

Exclusion criteria: Biopsies that were not processed, not fixed properly and with inadequate tissue structure were not included, malignant neoplasms were not included.

Preparation of Kumkum Solution

The maceration technique was used to prepare Kumkum solution. Fifteen grams of commercial Kumkum was diluted (Gopuram brand) with 100 mL propan-2-ol. The solutions are kept undisturbed for 48 hours. Filtered solution was used for staining, and the filtrates from those filters were kept in bottles with labels (Table/Fig 1).

Two sections of formalin-fixed paraffin embedded tissue measuring 5 μm thick were cut. The routine H&E staining was used in the first set. The prepared Kumkum stain was used for the second set of slides. Every batch was verified by using quality control slide and the timing of Kumkum solution was standardised to 4-5 minutes by trial and error method. A pH metre was used to calculate pH levels. A pH level of 5.45-5.52 was maintained in Kumkum solution. Two pathologists who were blinded independently evaluated the staining characteristics of both the groups using a scoring system (8):

1. Cellular architecture (CA):
Score-0: Indistinct nucleus- cytoplasm
Score-1: Distinct Nucleus- cytoplasm
2. Quality of Staining (QS)
Score-0=Poor
Score-1=Satisfactory
Score-2=Good

Steps in staining of Haematoxylin and Kumkum (H&K):

• Deparaffinise section followed by three changes of xylene five minutes each.
• Re-hydrate in absolute alcohol for five minutes.
• 70% alcohol for two minutes.
• Wash in running tap water for five minutes.
• Haematoxylin solution for eight minutes.
• Wash in running tap water for three minutes.
• 1% Acid alcohol one dip.
• Wash in running tap water for three minutes.
• 1% Lithium carbonate.
• Wash in running water for five minutes.
• Kumkum solution for four minutes.
• Rinse in 95% alcohol- 10 dips.
• Blot and mount.

Statistical Analysis

Results were tabulated and statistically analysed using chi-square test using SSPSS software (version 25).

Results

Out of 100 slides, 45 (90%) stained with H&E and 48 (96%) with H&K, showed distinct CA. A total of 47 slides (94%) stained with H&K and 41 (82%) with H&E showed good quality of staining (Table/Fig 2).

By crosstabulation of scores obtained from scoring of CA by Observer-1 and Observer-2 in H&E (Table/Fig 3) and H&K slides (Table/Fig 4), it was found that the Kappa value was 0.540 and 0.658 respectively, p-value was 0.001 both of which found to be statistically significant. Similarly, cross tabulating the scores obtained from scoring of quality of staining by Observer-1 and Observer-2 in H&E (Table/Fig 5) and H&K slides (Table/Fig 6), it was found that the Kappa value was 1.000 and 0.826, respectively, p-value of 0.001 both of which were found to be statistically significant.

Staining Characteristics of Different Cells of Kumkum

Squamous epithelium: The ectocervix lined by squamous epithelium was taken to compare. Kumkum staining highlighted all the layers of the epithelium clearly as eosin (Table/Fig 7).

Glandular epithelium: The long and tubular endometrial glands took the bright stain with blue nucleus. At places the subnuclear vacuolation can also be appreciated well (Table/Fig 8).

Erythrocytes: Erythrocytes were seen as bright red round to oval structures. In comparison to the surrounding tissue, it displayed excellent contrast. In fact, it appeared better in many cases compared to eosin section (Table/Fig 9).

Elastic fibers: Elastic Fibers in the artery were highlighted with dull pink fibers in connective tissue (Table/Fig 10).

Nerve tissue: Nerve tissue showed wavy nucleus with light pink colour.

Smooth muscle tissue: Smooth muscle tissue of Leiomyoma showed bundles and fascicles of dark pinkish red spindle-shaped cells with moderate cytoplasm (Table/Fig 11).

Discussion

Stains are a crucial component of laboratory procedure. Synthetic dyes are utilised for the majority of the stain in histology. Although synthetic colours are frequently effective, they can be harmful and cause allergy reactions to some (9). In India, kumkum is a powder with a red colour that is utilised for spiritual and religious purposes. Traditionally, it is made from Crocus sativus L. flowers and lightly spiced with turmeric (10).

The inherent absorption characteristics of distinct tissues with the stain are because of the ionic bond which exists between the dye and the tissue component. Factors playing an important role in this process are the pH of the solution, concentration of the dye and its time of action (11). In this study, the pH was maintained similar to that of eosin and time was standardised by trial and error method. The Kumkum solution was prepared using the maceration technique. Abraham M et al., discovered that when using natural dyes, sections stained using the maceration method yielded better and statistically significant results than sections stained using the soxhlet method, where the vapor of the natural extract is collected (12).

In histopathology, kumkum has the double property to stain both nucleus and cytoplasm for a variety of soft and hard tissues. In a study by Lavanya A et al., Kumkum staining was used to differentiate various components of oral tissue from bones to soft tissue. The author also highlighted that the incremetal lines displayed superior contrast in kumkum staining and as a result they might be used in forensic science. This kumkum stain can be used to differentiate between mature and immature bone and can be used to identify bone diseases such fibro-osseous lesions and neoplasms and to evaluate the stages of bone remodelling (6). Navy N et al., compared the staining characteristics of turmeric and Kumkum sections with those of conventional H&E in 57 cervical tissues and concluded that in H&K stained slides, the cytoplasmic and nuclear features were better appreciated than H&T and p-value was statistically significant (7).

In our study, connective tissue components such red blood cells, muscle fibres and collagen showed higher contrast in soft tissues slides stained with Kumkum similar to a Nigerian study. This finding has implications for collagen and muscular illnesses (13).

It can also be used in the Pap staining. Cytoplasm showed green to yellow hue of varying intensity depending on the degree of maturation of the cell. Nucleus which can be measured by animage software, appeared greenish-black coloured, but some showed yellowish tinge. Superficial cells showed light pink (14). Thus, it can be used in routine pap staining also in rapid staining like Diff Quick staining method (15).

Gupta A et al., also yielded a positive outcome of using kumkum. The staining procedure produced reddish-pink stained cells highlighted against the white background (16).

The commercially available Kumkum powder composition varies with each brand. But in general, it has components like turmeric powder, chalk powder, calcium salts and saffron. Kumkum which is prepared from the saffron flowers of Crocus sativus L. contains 150 volatile compounds and many non volatile compounds. The characteristic components of saffron which includes crocin (responsible for colour), picrocin (responsible for bitter taste) and safranal (responsible for odour and aroma). Saffron has been used as a histological colourant to stain connective tissue (17). Curcumin has tannins and flavonoids that imparts a brightened tinge (18). Artifacts due to stain precipitates can be avoided by filtration each time before staining. Some authors have suggested to include microwave heat technique to increase the general staining crispness (19). More studies must be done to establish its utility in staining malignant cells.

The cost of 25 grams of eosin stain is around 300 in Indian national rupees whereas that of 25 grams of kumkum powder is around 75 in Indian national rupees. Therefore, Kumkum stain appears to be a cost-effective alternate to eosin stain.

Limitation(s)

One of the limitations is that the durability of the stained Kumkum slides was not studied. The slides reviewed after three months showed preserved cell characteristics. Other limitation is only normal tissue architectures are compared, benign and malignant neoplasms are not included.

Conclusion

Kumkum solution appears to be an efficient counterstain in place of eosin in high lightening the normal structures in histopathological sections. It proves to be a cost-effective surrogate. It stained the RBCs in the arteries and cytoplasm of the cells in glandular and squamous epithelium better than or equally good as eosin. However, its utility in demonstrating benign and malignant neoplasms in histopathology and durability of the stained slides have to be studied.

DOI and Others

DOI: 10.7860/JCDR/2023/60639.17770

Date of Submission: Oct 09, 2022
Date of Peer Review: Nov 22, 2022
Date of Acceptance: Apr 03, 2023
Date of Publishing: May 01, 2023

AUTHOR DECLARATION:
• Financial or Other Competing Interests: None
• Was Ethics Committee Approval obtained for this study? Yes
• Was informed consent obtained from the subjects involved in the study? No
• For any images presented appropriate consent has been obtained from the subjects. No

PLAGIARISM CHECKING METHODS:
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• iThenticate Software: Mar 30, 2023 (8%)

ETYMOLOGY: Author Origin