A 1‐min double embedding method for small tissue specimens preserves comedone histology and eliminates the need for punch biopsies

Abstract

Background

It is difficult to preserve the structure and microbial distribution inside comedonal plugs during routine processing.

Objective

The objective of this study is to determine the optimal method to preserve the comedonal corneum plug structure and inherent microorganisms thereby eliminating the need to perform punch biopsies in relevant studies.

Methods

Corneum plugs were extracted from comedones of acne vulgaris patients. Primary embedding using either a 2% agarose, 2% agar, 25% gelatin, or 2% agar + 2.5% gelatin solution was subsequently performed and the results compared. The specimens were then fixed, waxed, sectioned, and examined by light, fluorescence, and scanning electron microscopies to observe the structures and microorganisms within the plugs.

Results

Both the 25% gelatin and 2% agarose solutions successfully preserved the structural integrity of corneum plugs and the inherent microorganisms. When considering other factors such as thermostability, reusability, and convenience, the 25% gelatin solution was the superior choice among the four materials.

Conclusion

We report a simple and effective method for double embedding comedonal plugs and other small tissue specimens. The technique preserves the structure and microbial distribution in situ within comedonal corneum plugs, eliminates the need for punch biopsies. This method may also be applied to other tiny and fragile tissue specimens, thereby enabling a potentially wide array of future large‐scale investigations and alleviated patients’ pain.

1. INTRODUCTION

Acne vulgaris (acne hereafter) is characterized by comedones—corneum plugs obstruct the hair follicular infundibula and orifices. ¹ Corneum plugs are composed of hyper‐proliferated corneocytes, sebum‐derived lipids, and a variety of microorganisms. ² The microorganisms and their metabolites, which include toxins and degraded sebaceous lipids, subsequently induce an inflammatory process, which persists throughout the disease course. ³

To explore the mechanisms of acne pathogenesis, previous studies have employed punch biopsies to investigate the corneum plug structure and microbial localization within the comedones. ⁴ , ⁵ , ⁶ Fluorescence in situ hybridization or immunofluorescence microscopy was the most commonly used method to study these features. ⁴ , ⁵ Although this was regarded as the only method that sufficiently allowed the study of follicular microbial localization and distribution, this procedure carries several ethical limitations. ⁷ As a result, few punch biopsies are performed, thereby limiting the ability to conduct larger‐scale investigations. These limitations become especially relevant when considering facial biopsies, and although some investigators have replaced facial biopsies with those from the trunk, the adequacy of these samples in representing the face remains uncertain. ⁷

With the exception of a few horny cells remaining in the follicular lumen, corneum plugs can be completely removed from comedones using comedone extractors. ⁸ In our prior study, we used fluorescence to examine the microbial distribution of extracted corneum plugs. ⁹ As a result, the excellent availability and abundance of microorganisms within corneum plugs was well‐documented, rendering these structures promising for future histopathological and microbial distribution studies.

Due to the plugs’ small size and fragility (because of lack of connective tissue), it is difficult to preserve their structure and inherent microbial distribution throughout the embedding and sectioning processes. For example, as a result of the organic solvents used in conventional fixation, dehydration, waxing, and sectioning procedures, a significant portion of microorganisms is lost.

Several compounds, including nitrocellulose, celloidin, agarose, and gelatin, have previously been used alone or in combination to double embed small, fragile specimens. ¹⁰ , ¹¹ , ¹² , ¹³ In those studies, the specimen were usually fixed, waxed, and then were embedded in those compounds to be ready for subsequent sectioning. We tried those protocols to treat corneum plugs but failed in obtaining optimized slices because the plugs are so loose in nature (compared to other tissues) that they are usually disintegrated during the first step, namely, fixation. We hypothesized that protection of the corneum plugs may be possible if the specimens were first embedded into a “shell” (primary embedding) material prior to the conventional embedding process (secondary embedding). Ideally, this shell would remain permeable to fixing liquids while simultaneously resisting dissolution by organic solvents and heat. The aim of this study is to determine the material and methods that most effectively preserve the intact structure and microbial distribution in situ when the plugs are embedded and sectioned.

2. MATERIALS AND METHODS

2.1. Patients and corneum plugs

With approval of the hospital ethics committee (approval number: 2017‐009), four patients with mild‐to‐moderate acne were recruited in the outpatient setting at the Shanghai Skin Disease Hospital. This study was registered at the Chinese Clinical Trial Registry (number: ChiCTR‐CPC‐17012398). Written informed consent was obtained prior to corneum plug collection. The extracted corneum plugs were immediately stored below 4°C in a sterilized Eppendorf vial and then transferred to the laboratory. At least five corneum plugs of similar size and type (open or closed comedones) were collected from each patient.

2.2. Primary embedding

Four corneum plugs from each patient were then embedded by one of the following four materials: 2% agarose (Regular grade, Takara Bio Inc., Japan), 2% agar (Chembase, Japan), 25% gelatin (Sinopharm, Shanghai, China), or 2% agar + 2.5% gelatin. The concentration was determined by preliminary experimentation (refer to the Supplementary Material, S1). A fifth plug was used as an untreated control and thus did not undergo primary embedding.

Each of the four materials was mixed into distilled water and heated to specific temperatures (98°C for agarose and agar, 50°C for gelatin) to maintain its aqueous state within the test tubes.

A single corneum plug was then placed onto a clean glass slide and embedded according to the following steps (Figure 1). The material solution was dropped onto the plug. As the temperature was lowered, the solution jelled, and the plug became embedded within the material. A sterile scalpel was then used to remove excess jell from the plug. The plug was then wrapped by a transparent tape (3 M, Shanghai, China), which was tailored into a sharp‐ended shape to mark the vertical axis of the corneum plug. This was performed to indicate the appropriate direction for further treatment and sectioning. The wrapped primarily embedded plug was subsequently fixed in 4% neutral formaldehyde overnight and then was placed into a 75% ethyl alcohol solution.

FIGURE 1.

Primary embedding process for a corneum plug: (A) A drop of primary embedding liquid was placed on a glass slide containing the corneum plug; (B and C) the excess jelled material was trimmed; (D) The specimen was wrapped by tape as a marker for direction.

2.3. Secondary embedding and sectioning

The plugs were then dehydrated, paraffin waxed, and sectioned continuously into 4‐μm slices by normal procedures. The slices were placed on poly‐lysine‐coated glass slides, heated at 60°C for 2 h, and then dewaxed and rehydrated. Three slides of each plug were stained by hematoxylin‐eosin (HE) for examination by light microscopy, while other slides were subject to examination by fluorescence microscopy and scanning electron microscopy (SEM).

3. RESULTS

Twenty corneum plugs were obtained from four patients with comedonal acne vulgaris. Sixteen of the plugs were double embedded by four different materials, and the remaining four negative controls were handled without primary embedding.

Light microscopy and SEM confirmed that gelatin most effectively preserved the corneum plug structure throughout the embedding and sectioning processes (Figure 2). Further examinations revealed the gelatin “shell” did not interfere with the subsequent secondary embedding, sectioning, or staining processes of the specimens.

FIGURE 2.

Light and scanning electron microscopic examination of corneum plugs embedded by different primary embedding materials

As shown in Figure 2, the structures of corneocytes stacked tightly in 25% gelatin were well‐protected and consequently maintained their wall structure. Wall morphology was the same as that of other biopsy‐obtained specimens in previous studies. ² , ⁸ , ¹⁴ Moreover, the plugs still contained the microorganisms in situ, which were readily observed by light, fluorescence, and SEMs. Malassezia spp. is shown as an example (Figure 3).

FIGURE 3.

Primary embedding by 25% gelatin maintains the corneum plug microorganisms in situ: (A) Malassezia spp. (arrow) in a plug from an hematoxylin‐eosin (HE)‐stained open comedo; (B) microorganisms lost in the plug without primary embedding; (C) microorganisms maintained by primary embedding (arrows); (D) Scanning electron microscopy (SEM) view of Malassezia spp. (arrow) pseudo‐colored in purple. (B and C) stained by calcofluor white and examined under fluorescence microscopy (λ_excitation = 365 nm)

In contrast, images from the untreated controls, as well as the plugs primarily embedded by 2% agar and 2% agar + 2.5% gelatin, exhibited inconsistent morphologies and separation of the corneocyte walls. Some walls exhibited feathering, while others were nearly stripped, leading to the loss of vulnerable microorganisms (Figure 2). These results revealed that primary embedding with 2% agar and 2% agar + 2.5% gelatin prior to fixation did not adequately protect the intact structure of corneum plugs during the secondary embedding processes. The performance of 2% agarose was superior to 2% agar and 2% agar + 2.5% gelatin but was inferior to 25% gelatin.

Additional performance characteristics of the four materials are summarized in Table 1.

TABLE 1.

Performances of the four primary embedding materials

Material	Protection	Thermostability a	Welding b	Convenience c	Cost	Stainability d
(1) 2% agarose	+/‐	+	–	–	–	+
(2) 2% agar	–	–	–	+/‐	+	+
(3) 25% gelatin	+	++	++	+	+	+
(4) 2% agar + 2.5% gelatin	–	–	–	–	+	+

^a Thermostability: The ability of being resistant to 70°C after fixation by 4% formaldehyde.

^b Welding: The potential to avoid splitting if the material is used to embed the same specimen twice, as discussed later in the text.

^c Convenience: Whether the solid jelly of the material can be stored, melted, and reused in future studies if the working solution is not completely consumed in the current study.

^d Stainability: The darker the staining by HE, the higher the stainability.

4. DISCUSSION

In this experiment, we sought to compare the efficacy of four different embedding materials in preserving overall corneocyte wall structure and microbial organization in situ within corneum plugs of comedones from acne patients. We also developed a double embedding process in which the plugs were (1) first embedded into a material, (2) fixed, and then (3) secondarily embedded by paraffin. This process is different from previous reported ones in which the steps were: (1) the specimen were fixed, (2) primary embedded into a material, (3) secondarily embedded by paraffin. Among our tested materials, 25% gelatin most successfully protected the structural integrity and associated microorganisms of the corneum plugs. To a lesser extent, 2% agarose was able to protect the overall morphology of corneum plugs.

The 25% gelatin is the superior embedding material in our study for several reasons. First, the structure of corneum plugs embedded in agarose still displayed some loosening of the wall structure. Second, agarose is more resistant to higher temperatures than gelatin (melting points at 95°C and 40°C, respectively). As a result, gelatin is more user‐friendly. Yet another benefit of gelatin is that it can easily be reused if the entire embedding solution is not expended at once, whereas agarose cannot be reused since it does not melt when the jelly is heated again.

In addition, the specimen must be successfully embedded at first attempt when using agarose jelly. In brief, if the specimen is not completely embedded, another drop of agarose solution needs to be added. However, these two drops do not uniformly weld together but rather split along the drop interface when the sample is later soaked in formaldehyde and ethyl alcohol solutions. This phenomenon does not occur in gelatin (Supplementary Material, Figure S2).

Another advantage of gelatin is increased strength relative to agarose. When exposed to formaldehyde, gelatin undergoes crosslinking in a manner comparable to protein tissues. We found that crosslinked gelatin becomes elastic and does not melt when heated (supplementary material). Finally, although gelatin has a deeper color shade than agarose after HE staining, the difference in color does not affect histopathological outcomes due to clear and sharp bordering between the embedding and embedded materials.

Contrary to our expectations, low concentration gelatin (4% or 5%) mixed with agar (2% or 4%) did not work in our study as previous studies reported. ¹² , ¹³ This may be due to differing agar and gelatin characteristics, their suppliers, or specimen size. The specimen size in our study was less than one millimeter, whereas in previous reports, the specimen was relatively large. ¹² , ¹³ This finding suggests that it may be necessary to adjust the actual concentration of materials when the suppliers and models of the materials are different.

Our primary embedding step is simple and effective, requiring 1–2 min per specimen. By coupling this primary embedding method with the conventional embedding process (hence the name, “double embedding”), larger‐scale studies concerning early‐stage acne vulgaris pathogenesis become simple to perform. Since this method allows investigators to forego the traditional need for a punch biopsy, the pain and ethical challenges of the biopsy procedure are avoided altogether. We would like to name this method the “ice method” after the pseudonym of the first author of this study.

An additional benefit of this double embedding method is that it enables investigators to handle other small (<1 mm) and fragile cutaneous specimens. For example, we successfully applied this process to a single human pilosebaceous unit (Supplementary Material, Figure S3). This procedure was used in an additional study, exemplifying its ability to potentially improve the handling of other human and animal tiny tissue specimens, and thereby proving itself an effective method for other relevant histopathological studies. ¹⁵

CONFLICT OF INTEREST

The authors have no conflict of interest in this study.

Supporting information

Supporting Information

Xu D‐T, Zheng Y, Shi Y, Jin H, Liu W, Wang X‐L. A 1‐min double embedding method for small tissue specimens preserves comedone histology and eliminates the need for punch biopsies. Skin Res Technol. 2023;29:e13235. 10.1111/srt.13235

DATA AVAILABILITY STATEMENT

Data are available within the article or its Supplementary Materials.