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New double embedding technique for specimens of endoscopic submucosal dissection using agarose: comparison with other media
  1. Masataka Zozumi,
  2. Mayumi Nakai,
  3. Takashi Ito,
  4. Ikuo Matsuda,
  5. Hiroyuki Hao,
  6. Yoshitane Tsukamoto,
  7. Seiichi Hirota
  1. Department of Surgical Pathology, Hyogo College of Medicine, Nishinomiya, Japan
  1. Correspondence to Professor Seiichi Hirota, Department of Surgical Pathology, Hyogo College of Medicine, Mukogawacho 1-1, Nishinomiya, Hyogo 663-8501, Japan; hiros{at}hyo-med.ac.jp

Abstract

Aims Procedure of endoscopic submucosal dissection (ESD) has been introduced widely for treatment of early gastric cancers. For such specimens, accurate pathological diagnosis, especially concerning depth of the invasion and exposure to margins, is essential to decide on the necessity of additional treatment. Therefore, easy and reliable tissue-processing method for multiple cut specimens is needed. The authors report here a new double embedding technique for specimens of ESD.

Methods Formalin-fixed whole specimen was superficially wrapped by agarose (the first embedding), and the tissue-agarose block was cut at 2–3 mm intervals. Each cut specimen was laid down with 90° rotation. This procedure permitted ‘on edge’ embedding of thin tissues in paraffin (the second embedding) and subsequent preparation of perpendicular section to the tissue surface. The authors compared the handleability and stainability among several media including various types of agar, agarose and gelatin for first embedding. A survey by questionnaire was carried out on handleability and/or impression on various tissue-processing steps from pathology technicians.

Results Among the media examined, agarose showed the best solubility in water and the best transparency on several representative stainings. According to the survey, pathology technicians seemed to feel that the present method was better than the usual tissue processing method, especially in shortened time consumption and accuracy of alignment of multiple tissues for ESD specimens.

Conclusions The present new double embedding technique using agarose provides not only an easy and reliable embedding procedure for technicians but also accurate and exact diagnosis for pathologists.

  • Double embedding technique
  • agarose
  • medium
  • endoscopic submucosal dissection
  • early gastric cancer
  • gastric cancer
  • methodology
  • morphology
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Introduction

Recently, early gastric cancers often have been treated by endoscopic procedure. Endoscopic mucosal resection may be applied only to small tumours (usually <20 mm in size), but endoscopic submucosal dissection (ESD) can be applicable to larger tumours. The ESD procedure makes it possible to avoid overtreatment, and can be carried out more easily, even in older patients or patients with various complications. Now, pathologists often encounter the diagnosis of such specimens, and the detailed pathological diagnosis including information on depth of the invasion and exposure to margins is essential for clinicians to judge the necessity of additional treatment. However, various problems concerning preparation of perpendicular and complete sections are not easy to resolve when the routine method of tissue processing is adopted.

In 1944, Samuel reported a technique using agar in embedding mammalian eggs.1 In the field of botany, Madge reported a technique of agar embedding in 1936.2

Later, double embedding method using agar was applied to small or slender human tissue, such as biopsy specimen or membranous tissue.3–8 Recently, a method was reported using a mixed agar–gelatin solution for the purpose of avoiding tissue shrinking during processing or a method utilising agarose for tissue microarray blocks.9 10 In 2008, the method of covering ESD specimens with agar and cutting tissues with agar was reported in the Japanese literature.11

Agar is a polysaccharide complex obtained through bleaching and hot water extraction from the red alga Rhodophyceae, and is composed of about 70% agarose and 30% agaropectin. Agarose is a neutral gelling fraction which consists of a linear polymer of alternating d-galactose and 3,6-anhydro-galactose units. Agaropectin is a non-gelling fraction which consists of β-1,3-glycosidically linked d-galactose units, some of which are sulfated.12 13 On the other hand, gelatin is a gelling substance of partially hydrolysed collagen extracted from the bones or connective tissues of animals such as cattle, pigs and horses.

The main purpose of the previously reported techniques using agar was to hold small or slender specimens in place without moving in the cassette or leaping away from the cassette during routine tissue processing. Those methods also made it easier to embed the tissue in accurate plane or to keep a proper orientation in small or slender specimens.14 15 Thus, the application of agar in tissue preparation has been limited to relatively small tissues so far. Now, we report a new double embedding technique for large ESD specimens using agarose. When various embedding media such as various types of agarose, various types of agar and gelatin were tested for comparison, agarose with a high gel strength was considered to be the best as a medium for the first-step embedding.

Materials and methods

Specimens and handling procedure

Early gastric cancer tissues were obtained by the ESD procedure and pinned on the cork board. Then, they were immersed in the formalin solution for fixation. The formalin-fixed tissues (figure 1A) were set in the plastic dishes of a suitable size (figure 1B). After dissolution with water using a microwave oven and cooling down to around 50°C, embedding medium was poured into the plastic dishes to cover the tissues (first embedding). When the embedding medium hardened, the tissues covered with the medium were removed from the plastic dishes. Then, unnecessary portions of the medium were trimmed off (figure 1C) and cut with 2–3 mm intervals perpendicular to the surface (figure 1D). The cut specimens were laid down with 90° rotation (figure 1E) and placed in plastic cassettes (figure 1F). After compressing them lightly with sponge piece, the tissues were processed to dehydration, xylene penetration, paraffin infiltration and paraffin embedding (second embedding, figure 1G). Sections were made by cutting paraffin blocks with a microtome.

Figure 1

Double embedding technique using 2% Agaraose MP for an endoscopic submucosal dissection (ESD) specimen. (A) Formalin-fixed ESD specimen of the stomach which had been pinned onto the cork board. (B) ESD specimen set in a plastic dish. (C) Tissue unified with agarose after extra-agarose portions were trimmed off. (D) Multiple cut specimens with medium. (E) Laid down specimens with 90° rotation. (F) Multiple cut specimens set in plastic cassettes. (G) Paraffin blocks of the multiple cut specimens. The scale bar on the right upper corner of each part means 1 cm length.

Media used for first embedding

We assessed various media (six types of agarose, six types of agar and one type of gelatin) listed in table 1 for first embedding. The concentration of the media was optimised at 2% after the preliminary examination.

Table 1

List of the embedding media

Various stainings, immunohistochemistry and evaluation of stainability

The sections were stained with H&E, Alcian blue, elastica van Gieson (EVG), periodic acid–Schiff (PAS), Victoria blue with haematoxylin–eosin (VBHE), Giemsa and Masson's trichrome. Immunohistochemistry for α-smooth muscle actin (DAKO, Glostrup, Denmark) and D2-40 (DAKO) was also performed, since these two antibodies are often used for diagnosis of ESD specimens with early gastric cancers. The concentration of the antibodies used was 1:100 dilution for α-smooth muscle actin and 1:50 dilution for D2-40, and ChemMate Envision Kit/HRP (DAB) System (DAKO) was used for the detection. The following graded system was employed for evaluation of the stainability of the media when observed by microscopy: – when the medium was completely invisible; ± when it was faintly visible; + when it was visible and similarly stained as compared with the adjacent gastric tissue; and ++ when it was visible and more strongly stained than the adjacent gastric tissue.

Questionnaire on handleability and/or impression on various tissue-processing steps for pathology technicians

The questionnaire included six items concerning embedding, sectioning and staining listed in box 1. A five-point graded system compared with an ordinary method (score 3) was used, and eight technicians in our department answered the questions.

Box 1 Questionnaire about handleability and/or impression on various tissue processing steps

  1. Questions on the embedding procedure

    • Q1. Time consumption for embedding

      • 1. long

      • 2. slightly long

      • 3. similar

      • 4. slightly short

      • 5. short

    • Q2. Accuracy of tissue alignment on embedding

      • 1. bad

      • 2. slightly bad

      • 3. similar

      • 4. slightly good

      • 5. good

    • Q3. Risk of orientation loss (rotation or malposition of specimens)

      • 1. high

      • 2. slightly high

      • 3. similar

      • 4. slightly low

      • 5. low

  2. Questions on the sectioning procedure

    • Q4. Achievement of flat surface of the paraffin blocks

      • 1. difficult

      • 2. slightly difficult

      • 3. similar

      • 4. slightly easy

      • 5. easy

    • Q5. General impression on sectioning with microtome

      • 1. bad

      • 2. slightly bad

      • 3. similar

      • 4. slightly good

      • 5. good

  3. Question on the staining procedure

    • Q6. General impression on staining

      • 1. bad

      • 2. slightly bad

      • 3. similar

      • 4. slightly good

      • 5. good

Statistical analysis

Results from the questionnaire were analysed using a Wilcoxon signed-rank test. Differences were regarded as significant when a score was <0.05.

Results

Comparison on first embedding and cut steps among embedding media used

All types of agarose showed a better solubility in water using a microwave oven than all types of agar and gelatin. Regarding strength and elasticity of the media, all types of agarose except NuSieve GTG agarose and all types of agar except purified Agar were sufficient. However, NuSieve GTG agarose, purified Agar and gelatin were too fragile to be grasped by forceps or cut by a blade. Concerning the transparency of the gelled media, none of them disturbed observation of surface of the ESD specimens through the medium.

Comparison on paraffin-embedding step among embedding media used

All types of agarose and agar did not affect the manipulation of paraffin embedding. Since the specimens unified with media which were dehydrated and paraffin-infiltrated were thick enough to be placed perpendicular to the tissue surface in order, the paraffin-embedding step in medium-using tissue processing appeared to be generally easier than that in ordinary tissue processing.

Comparison on stainability of media and tissues among embedding media used

The first embedding media were often coloured at various degrees in various staining methods. In H&E staining, all types of agar and gelatin were visible as a pale, reddish and amorphous substance (figure 2). On the other hand, all types of agarose were almost completely invisible (figure 2). Similarly, all types of agarose were not visible, but all types of agar and gelatin were visible in Giemsa staining (figure 2). In PAS staining, all types of agarose, all types of agar and gelatin were visible (figure 2). In Alcian blue, EVG and VBHE stainings, all types of agarose were visible or faintly visible, but all types of agar and a type of gelatin were more clearly visible than agarose (figure 2). In Masson's trichrome staining, all media used were invisible (data not shown). Immunohistochemistry for α-smooth muscle actin and D2-40 gave no staining in all types of media used (figure 2). The results are summarised in table 2.

Figure 2

Representative results of various stainings using Agarose MP and Bacto Agar. Agarose MP showed no apparent staining in haematoxylin–eosin (HE) and Giemsa stainings. In Alcian blue, elastica van Gieson (EVG), periodic acid–Schiff (PAS) and Victoria blue with H&E (VBHE) stainings, Agarose MP was visible or faintly visible. On the other hand, Bacto Agar was generally more clearly visible than Agarose MP. Immunohistochemistry for α-smooth muscle actin (SMA) and D2-40 did not lead to staining in either Agarose MP and Bacto Agar (arrow: no medium area).

Table 2

Summary of stainability of various types of media

On the other hand, the histology of the tissue specimens in all media used did not seem to be affected by double embedding tissue processing in all staining methods tested compared with ordinary tissue processing. The gastric cancer cells in the mucosa were easily recognisable on various staining methods, as shown in figure 3.

Figure 3

Influence of the double embedding technique using agarose on the diagnosis of early gastric cancer. Infiltrating adenocarcinoma cells in the mucosa in two cases (A, B) were not affected by the embedding technique in H&E staining. In various staining methods on serial sections of another case (C, H&E; D, elastica van Gieson; E, D2-40 immunohistochemistry; F, α-smooth muscle actin immunohistochemistry), each method clearly stained an objective structure, and cancer cells were easily identified. The scale bar on the upper right corner of each part denotes 100 μm.

Assessment of questionnaire for pathology technicians

In questions on the embedding procedure (Q1 to Q3), all three items showed significantly higher points when compared with the ordinary procedure (Q1, 4.4 vs 3, p=0.03; Q2, 5 vs 3, p=0.0156; Q3, 4.57 vs 3, p=0.0156). In questions on the sectioning procedure (Q4 and Q5), two items received higher average points but did not show statistical significance when compared with the ordinary procedure. In the question on the staining procedure (Q6), one item did not show a higher point.

Discussion

We have reported here a new method of double embedding technique for ESD specimens using agarose. We compared the handleability and stainability among several media including various types of agarose, agar and gelatin for the first embedding step, and found that agarose generally showed the easiest handleability on dissolution in water and the best transparency on observation by microscopy among the media examined.

In the past, agar was usually used as a medium to hold small tissues in place because they are easily rotated and difficult to embed in an accurate plane during routine tissue processing. In the present method, the double embedding technique was applied to larger tissues obtained by the ESD procedure.

In recent Japanese literature,11 a similar double embedding method for ESD specimens using agar was reported. The basic procedure of their method and the present method were the same at the points of wrapping specimens by medium, cutting them with medium and processing to embedding. In the present study, we compared the handleability and stainability among several media including various types of agar, agarose and gelatin, and found that agarose with high gel strength is most suitable for the first embedding medium among the media examined.

ESD procedure has been widely applied to early gastric cancers, and now pathologists often diagnose those cases. However, pathological diagnosis especially concerning depth of invasion and exposure to margins is not always definitive because it is not easy to embed all of the multiple sections in an accurate order and accurate ‘on edge’ position in usual tissue processing. The present method appeared to show better results in those views than the method of usual tissue processing did.

According to the questionnaire on tissue processing for pathology technicians, they seemed to feel that the present method was generally better than the usual tissue processing method for ESD specimens. In particular, shortened time consumption, accuracy of alignment of multiple cut tissues and decreased risk of losing orientation are considered to be better points.

We used the present method for ESD specimens, but this could be applicable to other specimens. Fragile and papillary-shaped colon polyps seem to be suitable for this method, and thin membranous tissues such as large cystic walls may also be candidates for the method. Very recently, we found that the specimens unified with agarose after dehydration and paraffin infiltration were easier to cut thinner than those before dehydration and paraffin infiltration. It is possible to cut tissues even in 1 mm thickness. This procedure may improve the present double embedding method, and the possibility is under consideration.

In conclusion, the present double embedding technique using agarose is applicable to large tissues such as ESD specimens, gives accurate ‘on edge’ embedding and exact diagnosis of the specimens, and minimally affects observation by microscopy.

Take-home messages

  • Our study confirmed that the double embedding technique can be applied to large tissues obtained by ESD procedure.

  • As a first embedding medium, agarose with high gel strength was the most suitable.

  • This technique provides not only an easy and reliable embedding procedure for technicians but also an accurate and exact diagnosis for pathologists.

Acknowledgments

We thank our Pathology technicians (M Oda, R Hariyama, Y Okamura, M Itoyama, R Enomoto, J Nakamura, G Sato and M Ishida) for their technical assistance and cooperation in the questionnaire.

References

View Abstract

Footnotes

  • Competing interests None.

  • Patient consent Obtained.

  • Provenance and peer review Not commissioned; externally peer reviewed.

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