Ideal and excellent tissue orientation is the most crucial step in tissue processing to demonstrate proper morphology in the sections, which aids in definite diagnosis [1]. Although paraffin is the most widely used material for embedding, however in some occasions, its necessities minor alternatives or modifications to produce significant results. Most of the oral pathologists would have faced problems in handling small/thin /delicate tissue samples, especially in cases of multiple small incisional tissues, where tissues are tough to process and orient properly due to further tissue shrinkage during processing [2, 3].

Pulp tissue section can be used for various educational and research purpose. But, preparing the section of pulp tissue alone is the foremost difficult task owing to its small and delicate nature that often results in many folds during orientation and processing [4]. Improper orientation leads to re-orientation of the tissue which is an unacceptable waste of time. This problem can be resolved by surrounding the samples with a material that will keep them in the desired position during the entire procedure. Despite many advanced techniques followed in pathology nowadays, all these techniques are based on basic and classical methods used in the past. The double embedding technique using agar seems to be one of the classical methods illustrated in past editions of surgical pathology manuals [5, 6]. V.B. Wigglesworth used the embedded agar—supported tissues and infiltrated in ester wax and reported successful sectioning of 0.5—1μm range of an insect cuticle [7]. Later G.R. Buzzell modified and used this technique both for ester wax and paraffin [8].

Over the years, agar-paraffin technique has been widely used for small or thin biopsies such as corneal, conjunctiva, buccal mucosal, vocal cord, temporal artery biopsies, endo-bronchial biopsies, to get the combined results of both embedding medium [9, 10]. Though agar-paraffin technique method produces excellent and significant results in pathological sections, yet it is of minimally used technique in oral pathology laboratory. Hence, the present study was aimed to evaluate and compare the ease of embedding and sectioning using Agar-Paraffin double embedding technique for small/minute oral mucosal biopsies and pulp tissues with that of the blocks prepared using the routine paraffin technique. To best of our knowledge, this is the first study to use double embedding technique for pulp tissues.

Material and methods

The present study was carried out at the KSR Institute of Dental Science and Research, Tamil Nadu, India. Ethical Clearance from the Institutional review board was obtained prior to the study. A total of 40 oral tissue samples categorized into two groups were taken for this study. Group I included 20 archival oral mucosal biopsies (soft tissue) samples of size ranging from 0.2 to 0.5 cm, and Group II included 20 pulp tissues obtained from a freshly extracted non-carious tooth extracted for orthodontic and Prosthodontic purposes. In each group, ten blocks were prepared by the routine paraffin embedding method, and 10 blocks were prepared by agar-paraffin double embedding method.

Preparation of agar-paraffin double embedded blocks

First, the embedding surface of the tissue was identified and marked by the colouring ink (fevicryl acrylic hobby colour) (fig. 1, a) and kept dry on filter paper. Then, 3% agar was prepared by dissolving Type I agar powder (bacteriological agar) in 10 ml of distilled water and gradually brings it to boil on a hot plate with continuous stirring. This solution was allowed to cool to room temperature. The tissue was placed on the glass slide with its marked embedding surface facing down, and then the molten agar was poured over it (fig. 1, b). Agar solidifies in 8 — 10 min, and excess was trimmed so that 3 mm of agar was left all around the tissue. The pulp tissues were also pre-embedded in agar in the same manner without any folds. These agar blocks were processed following the routine procedure. After it gets processed, impregnated, the agar block containing the inked biopsies was ready for embedding with its marked embedding surface facing down into the molten paraffin wax. Thus, the double embedded agar-paraffin blocks for soft tissue and pulp tissue were ready for routine sectioning (fig. 2) and all the prepared slides were stained with H&E. All the stained slides were evaluated.

Fig. 1. Steps of preparation of agar-paraffin double embedded blocks.

a — the embedding surface of the tissue was identified and marked by the colouring ink (fevicryl acrylic hobby colour); b — cooled agar was poured over the tissues placed with the marked embedding surface facing down on a glass slide.

Fig. 2. Prepared agar blocks.

a — soft tissue agar blocks; b — pulp tissue agar block.

Preparation of paraffin embedded blocks

Paraffin blocks were prepared from the remaining 10 pulp and 10 tissue samples in both the groups following the routine procedure and were stained with H&E for standard comparison.

Scoring criteria and evaluation

The samples were evaluated for the following features such as: ease of embedding and ease of sectioning. All tissue blocks were prepared and sectioned using soft tissue microtome. The ease of embedding was evaluated by observing the time taken to embed/orient the tissue/agar block in the molten paraffin wax. The ease of sectioning was evaluated by the quality of the sections like proper ribboning without folds, whether it can cut the entire tissue without dislocation it from the block, ease of getting sections onto the glass sides.

Scores were given by comparing all these criteria with the routine paraffin embedding technique as excellent — 3, good — 2, fair — 1 and inadequate — 0. Scores obtained in both the techniques for pulp and tissue samples were analysed using the chi-square test, and it was compared between the techniques and the samples (pulp and oral soft tissues).

Results

Ease of embedding. On evaluating the ease of embedding the soft tissue between the two groups, mean values were higher for embedding done using agar (2.9±0.31622) than the mean value for embedding done using paraffin (1.8±0.42163) (table 1) and it also showed a statistically significant result (p=0.000) (table 2).

Table 1. Mean values obtained in groups

Table 2. Ease of embedding soft tissue in agar vs ease of embedding soft tissue in paraffin — group I

Note. * — p<0.05 — Significant.

On evaluating the ease of embedding the pulp tissue between the two groups, mean values were higher for embedding done using agar (3.0±0.0) than the mean value for embedding done using paraffin (1.7±0.67464) (see table 1) and it showed a statistically significant result (p=0.000) (table 3).

Table 3. Ease of embedding pulp tissue in agar vs ease of embedding pulp tissue in paraffin — group II

Note. * — p<0.05 — Significant.

Ease of sectioning. On evaluating the ease of sectioning the soft tissue between the two groups, mean values were higher for sectioning done using paraffin (2.6±0.51639) than the mean value for sectioning done using agar (2.0±0.6667) (see table 1) and it showed a statistically significant result (p=0.041) (table 4).

Table 4. Ease of sectioning soft tissue in agar vs ease of sectioning soft tissue in paraffin — group I

Note. * — p<0.05 — Significant.

On evaluating the ease of sectioning the pulp tissue between the two groups, mean values for sectioning done using paraffin (2.0±0.6667 and mean value for sectioning done using agar (2.0±0.6667) (see table 1) and the result was not statistically significant result (p=1.000) (table 5).

Table 5. Ease of sectioning pulp tissue in agar vs ease of sectioning pulp tissue in paraffin — group II

Note. * — p<0.05 — Significant.

Intergroup comparison of the mean values obtained for embedding and sectioning is shown in the fig. 3.

Fig. 3. Intergroup comparison of the mean values obtained for embedding and sectioning.

Discussion

Embedding is the process which involves enclosing of properly processed, correctly oriented specimens in a medium that provides external support during processing [11].

The bacteriological agar is made up of galactose subunits extracted from the cell wall of red algae [12]. Because of its property of hysteresis, it is used chiefly for tissue embedding, and it remains solid at 36±1.5 °C, continues to remain firm at 60—65 °C (when paraffin wax is molten, thus holding the embedded tissue tight and well oriented) and melting temperature of 87±1.5 °C, a temperature range, which a tissue processor never reaches [13]. Thus, the agar remains solid throughout the tissue processing, and the embedded oriented tissue fragment in agar remains secured and oriented all through the process. Another advantage was that, during processing, agar does not hinder the penetration of alcohol into tissues embedded in it [14].

Conventionally, it is always difficult to orient minute tissues as small as 0.2 cm because of the reduction of the tissue volume and surface area to near half of the original size after formalin fixation and paraffin processing [4, 13]. In such situations, it is difficult to identify the embedding surface, in this present study, agar-paraffin embedding technique has overcome this problem, because we have pre-embedded the tissues in agar, and as the agar is colourless and transparent in liquid, gel and solid forms; the orientation of these agar blocks can be easily viewed/monitored with the naked eye and embedded in paraffin [14, 15].

In the present study, mean values were higher for embedding done using agar (2.9±0.31622) than the mean value for embedding done using paraffin (1.8±0.42163) and it showed a statistically significant result (p=0.000) which is in accordance with the study done by S. Sandhya et al [11].

In the present study, thus, this technique of agar-paraffin embedding facilitates tissue fixation, processing and optimal orientation and avoids tissue loss.

However, agar paraffin blocks were easy to do sectioning with adequate ribbon formation (without tears) (fig. 4). No technical difficulties were experienced while sectioning which result in quality sections. The sections were easy to spread as the agar did not shrink appreciably during processing and were least subjected for folds and because of smooth cut, resulted in no holes/chatters. The coloured inks remained permanently on the tissues in the blocks. This helped in the easy identification of tissue in a block and helped to make sure all the fragments were trimmed well to get complete sections [16].

Fig. 4. Adequate ribbon formation (without tears) during tissue sectioning.

Once the sections were cut and made float in a 50—55 °C water bath for mounting on to the marked glass slides, the agar became soft, allowing the tissue to expand as required for proper mounting onto glass slides with minimal or no folds, especially for the pulp tissues [17]. Jones and Calabresi used gelatin with agar which allows re-expansion and results in tissue section without folds. Agar persists around the sections and minimally stained with a faint background (fig. 5, 6). However, this will not interfere with the tissue analysis because the tissue itself is not infiltrated by agar, as it only acts as a pre-embedding medium [13]. Based on the overall observations in our study, this improvised double embedding technique is best suited for embedding thin tissues (pulp tissue) & minute biopsies efficiently. It also showed equal sectioning efficiency as that of the paraffin blocks. However, additional experience may be needed to validate it further.

Fig. 5. Soft tissue sections of different embedded blocks.

a — paraffin embedded tissue section; b — agar embedded tissue section. H&E, ×10.

Fig. 6. Pulp tissue sections of different embedded blocks.

a — paraffin embedded pulp tissue section; b — agar embedded pulp tissue section. H&E, ×10.

Conclusion

The modified agar-paraffin embedding technique represents a simple, reliable, user-friendly method that can significantly improve the quality of diagnostic information one can obtain from minute biopsies and pulp tissues by improving tissue orientation and quality of sections. In the present study, though no statistically significant difference was seen among embedding and sectioning sections between the two groups, the average ease score for the double embedding of minute biopsies showed better scores than the pulp tissue with that of the routine technique. Thus, we would like to conclude this method as a reliable and straightforward alternative technique that helps in better orientation, processing and sectioning, especially for oral small or thin biopsies.