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Year : 2015  |  Volume : 58  |  Issue : 1  |  Page : 12-16
Improvised double-embedding technique of minute biopsies: A mega boon to histopathology laboratory

Department of Pathology, St. John's Medical College and Hospital, St. John's National Academy of Health Sciences, Bengaluru, Karnataka, India

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Date of Web Publication11-Feb-2015


Introduction: Optimal orientation of minute mucosal biopsies is essential for a definite diagnosis in gastrointestinal pathology or to visualize neural plexuses in Hirschsprung disease. The problem of minute size of the biopsy and its orientation gets compounded when they are from neonates and mandates exhaustive strip cuts, thus delaying reporting. Aim: A modified agar-paraffin technique is aimed to make tissue embedding efficient and user-friendly by inking mapping biopsies (one or more) either fresh or fixed with surgical coloring inks followed by embedding first in agar after orientation and followed thereafter by processing, re-embedding in paraffin wax, sectioning and staining. Results: The tissues in agar paraffin block were found to be well processed, firm, held secure and well preserved. The blocks were easy to cut, with serial sections of thickness 2-3 μ and easy to spread. The colored inks remained permanently on the tissues both in the block as well as on the sections which helped in easy identification of tissues. Agar did not interfere with any stain such as Hematoxylin and Eosin or with histochemical stains, enzyme histochemistry or immunohistochemistry. Inking biopsies and pooling them in a block when obtained from the same patient reduced the number of tissue blocks. Conclusion: The modified agar-paraffin embedding technique is a simple reliable user friendly method that can greatly improve the quality of diagnostic information from minute biopsies by optimal orientation, better quality of sections, faster turnaround time and cost-effectiveness by economizing on the number of paraffin blocks, manpower, chemical reagents and laboratory infrastructure.

Keywords: Agar-paraffin block, coloring inks, Hirschsprung disease, mucosal biopsies

How to cite this article:
Yadav L, Thomas S, Kini U. Improvised double-embedding technique of minute biopsies: A mega boon to histopathology laboratory. Indian J Pathol Microbiol 2015;58:12-6

How to cite this URL:
Yadav L, Thomas S, Kini U. Improvised double-embedding technique of minute biopsies: A mega boon to histopathology laboratory. Indian J Pathol Microbiol [serial online] 2015 [cited 2019 Jan 23];58:12-6. Available from:

   Introduction Top

The optimal orientation of minute biopsies during paraffin block embedding facilitate visualization of the mucosal architecture in assessing villous morphology in malabsorption syndrome or neural plexii in well-defined tissue planes for definite tissue diagnosis of Hirschsprung disease (HD). The difficulty is amplified further when these minute biopsies are from neonates. In gastroenterological units, the mucosal biopsies are placed on thin cut cardboard/thick paper pieces with mucosa facing upwards. This latter technique greatly facilitates orientation and is widely practiced. [1] Yet, bowel biopsies in suspect aganglionosis often reach the laboratory irregularly curled up, both when sent fresh in saline or in formalin. The time spent in reorientation is a waste of the pathologist's time and energy.

Regardless of the state on reaching the laboratory (fresh/fixed) or the fixatives used, it is necessary to precisely orient mucosal biopsies. The most suitable material for tissue embedding is the bacteriological agar. [2],[3] We aimed to evaluate a modified technique of agar embedding of minute biopsies. We have examined a method of orienting and embedding fresh tissue initially in optimal cutting temperature (OCT) medium for frozen section to demonstrate acetyl cholinesterase activity and thereafter re-embedded the frozen tissue remains in agar. In tissue sent in a fixative like formalin, we have embedded first in agar before further tissue processing and paraffin wax embedding. This technique of agar embedding of minute biopsies seems simple, efficient and user-friendly.

   Materials and Methods Top

Mucosal biopsies, received fresh or fixed in formalin, were examined with a magnifying glass to orient them and their measurements in greatest and smallest dimensions noted. The embedding surfaces were identified by noting the velvety, pale pink mucosa and the adjacent hemorrhagic submucosa. These were kept dry, oriented perpendicular to the cutting surface on a filter paper and that surface was marked gently by surgical coloring inks from Histolab/acrylic paint from Fevicryl acrylic hobby color groups (no. 6, 12, 22, 33) [4] using tip of a paper strip. Seromuscular bowel biopsy was identified as a C-shaped pale, translucent firm structure; this 'c' was considered as the embedding surface. This is a very important step in biopsy orientation as practiced during frozen section and the C-shape of the fragment occurring due to contracture of smooth muscle especially the muscularis propria, is taken advantage for the proper orientation of mucosal biopsies with attached muscle, especially when the fragment is not sent fixed on paper. When leveling biopsies were received from various sites of the colon, that closest to the anus was colored green, followed by yellow, blue and India ink sequentially. Thus, more than one colored fresh tissue was embedded in OCT medium in one block [[Figure 1]a], mimicking a microarray block for cutting frozen sections after documenting the color against the origin of the tissue sample. The cut frozen sections were stained with rapid Hematoxylin and Eosin (H and E) as well as processed for enzyme histochemistry. After the frozen sections were reported, the OCT embedding medium was allowed to thaw; the inked tissue washed lightly with water to take away OCT remains and re-embedded in the next medium, namely agar as described below [[Figure 1]a-c].
Figure 1: (a) Minute biopsies colored with different inks embedded in freezing medium for frozen section, (b) Biopsies colored with different inks embedded in a single block of agar-paraffin and its corresponding section stained with routine (H and E), (c) Set of materials required for agar embedding technique. Inset: Colored biopsies embedded in agar medium

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3% of bacteriological agar (SISCO Research Lab, extra pure bactograde Lab Reagent) in 10 mL of distilled water was prepared in a beaker by gradually bringing to boil on a hot plate with continuous stirring. This solution was slowly cooled to room temperature and poured into the steel embedding moulds. To hasten the gelling process, the moulds were kept in the freezer/cryostat for 1-2 min. If not, the agar solidifies in about 8-10 min at room temperature. When it is solidified, four punch holes were made for the four biopsies in one block using punch biopsy forceps of diameter 1-6 mm depending on the size of the biopsy. The inked biopsies which were already oriented on the filter paper were gently lifted with fine forceps and placed into the punch holes in the agar block with their embedding surface facing down. The other three biopsies were similarly embedded in the agar sequentially. Molten agar was poured on top of the tissue in the punch hole to fill the cavity and seal it [[Figure 1]b, inset].

After the agar was set, the agar block containing the embedded tissue was easily detached from the embedding moulds and the edges cut to ensure that 3 mm of agar was left all around the tissue. All the tissue samples thus embedded were in a single plane, that is, the embedding surface of the agar block was the embedding plane for all the tissues. Subsequently, this agar block was placed inside a disposable cassette and processed in a histokinette using the regular cycle used for other tissues. After the last phase of impregnation, the agar block containing the four different colored tissues was ready for embedding with their embedding surfaces facing down into the molten paraffin. This double preembedded agar-paraffin block [[Figure 1]c] was allowed to cool on a cold plate after which it was ready for serial sectioning at 4 μ. They were cut in the same manner as the routine paraffin embedded blocks. The sections, thus, obtained were either stained with H and E [[Figure 1]c and [Figure 2]a] or taken on coated slides for immunohistochemistry.
Figure 2: (a) H and E, section (×40) from blue inked biopsy embedded in agar-paraffin. The blue ink has not interfered with the (H and E) (b) AB-PAS section from blue inked biopsy shows AB-PAS positive glands. The blue ink has not interfered with the special stain (×40) (c) AChE histochemistry on rectal mucosa in Hirschsprung disease shows increased activity (black fibres). The yellow ink has not interfered with the enzyme activity (×100) (d) Rectal biopsy (colored red) embedded in agar and stained for calretinin highlights ganglion cells in myenteric plexus. The red ink has not interfered with the immunostain (×400)

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We have also processed other biopsy samples (e.g., corneal discs after penetrating keratoplasty, endobronchial biopsies, soft tissues biopsies, core biopsies, circumferential ring bowel biopsies etc.) similarly.

At the end of the technique, all the biopsies were evaluated for the following features:

  • Ease of embedding and cutting sections.
  • Degree of tissue processing.
  • Numbers of paraffin blocks used.
  • Microscopic tissue orientation and least number of sections necessary for proper assessment.
  • Interference of coloring inks in the gross and microscopic interpretation.

   Results Top

One hundred and two mucosal biopsies from sites such as colon, cornea, conjunctiva, vocal cord and bronchus ranging in size from 0.2 to 0.5 cm were processed by this improvised double embedding technique over a study period of 2 months in the histopathology laboratory attached to a tertiary teaching medical college hospital. The technique went through a series of improvisations till it was well standardized, made user friendly and acceptable by technical staff and yielded excellent results to be employed on a routine basis in the laboratory.

Conventionally, it is always difficult to orient minute mucosal tissues as small as 0.2 cm because of the reduction of the tissue volume and surface area to near half after formalin fixation and paraffin processing. In such situations, it is difficult to identify the embedding surface during embedding as the coloring inks invariably color the entire tissue and not the embedding surface alone. The preembedded agar-paraffin blocking technique has overcome this issue as the tissue when fresh would have already been embedded in agar before processing. The results of this technique have been evaluated and are listed below:

Ease of embedding and cutting sections

The technique has helped in optimally orienting biopsies as small as 0.2 cm. We were able to embed easily such stained tissues in agar at a convenient time. They were processed along with the other routine tissue blocks and embedded with other blocks in paraffin by any technical staff. We did not need an experienced staff to do this exercise. As the agar was colorless and transparent in liquid, gel and solid forms, the orientation of the biopsy could be easily viewed/monitored with naked eye or through a dissection microscope and stained with coloring inks as applicable. The agar paraffin blocks were easy to cut and were well suited for cutting serial sections of thickness as thin as 2-3 μ. No technical difficulties were experienced while cutting 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, provided there were no trapped air bubbles while preparing the blocks. The colored inks remained permanently on the tissues in the blocks [[Figure 1]c]. This helped in easy identification of the various tissues especially when there were >1 tissue in a block and helped to make sure all the fragments were trimmed well so as to get complete sections.

We encountered one problem during orientation. If tissues were minute and were not held down during embedding in agar, they could float and come up to get placed themselves at different levels in the agar block making it difficult for all tissue fragments to be placed in a single plane. As a result, tissue fragments did not appear as complete cuts in a single section at any given time. In such situations, the block had to be cut carefully at different levels to get to have all tissue fragments; this resulted in more work, loss of time and undue delay. We tried to overcome this problem by gently holding down the tissue by mounting needle till the agar poured into the punch hole over the fragment solidified. When there were >1 tissue to be embedded, the tissue needed to be held down for every fragment. This care taken was worth the effort at the end of the exercise. This problem could also be overcome by embedding on a glass slide if the tissues are too small as described by Ventura et al. [5] The agar is poured on a glass slide and allowed to gel and then tissue is placed into the gel as described above. The process is faster by this method as the agar used is less. The thin plate of agar with inked tissues embedded in it on a glass slide as described above, is lifted up with a blade, edges cut and re-embedded in a larger agar block as described above. As the height of the agar block is thin and as the fragments inked can be easily visualized with ease, this improvisation was a great help to get the right plane for minute tissues during embedding.

Assessment of degree of tissue processing

These agar-paraffin embedded tissues were found to be well processed, firm and securely held and hence the agar as a preembedding medium for tissues inside had not interfered with the penetration of chemical reagents while processing. It was also noted that the tissues in the agar medium had shown minimal shrinkage in their sizes after processing and no loss at any time. In situations, were the fragmented biopsies were too small to be picked up, the fluid (saline/formalin) with the tissue fragments is spun at 1000 rpm for 30 s. The fluid is decanted, a drop of dye added to the sediment and the entire deposit is embedded in-toto into a well-made in the solidifying agar on a glass slide as discussed above. Thus, even the smallest fragment though not visible with naked eye in agar block was identifiable microscopically as the tissue was inked. The tissue in agar and paraffin block remained well preserved like any other routine paraffin block.

Number of blocks used

The number of blocks were cut down drastically during frozen as well as in the paraffin block depending on the type of biopsy received - fresh as for frozen or fixed in formalin. Differential gross coloring of tissue samples and group embedding in OCT medium for frozen section helped in many ways. Instead of cutting one frozen section for each tissue biopsy, one needed to cut only one block at one go with as many as four biopsies in it but colored differently, thus saving an easy 60 min of turnaround time (TAT) for frozen section (accepted TAT for one frozen block is 20 min as per National Accreditation Board for for Testing & Calibration of Laboratories (NABL)). We embedded a maximum of 4 minute biopsies colored differently in a single agar block without any difficulty provided the biopsies were small and >4 different colored inks were available.

Microscopic orientation and least number of sections necessary for proper assessment

Since the biopsies were preembedded in agar in a desired orientation and agar embedded tissues were found to be well processed, firm and securely held, the orientation remained as it is through the entire process till the microscopic study. This helped in avoiding deeper sections unnecessarily and therefore, also helped in assessing the biopsies with least number of sections. As agar-paraffin block mimicked a tissue microarray block, [5] one single section containing a maximum of four tissue samples from four different sites from the same patient helped in cutting down the number of blocks to be cut, manpower and reagents for special techniques such as enzyme histochemistry and immunohistochemistry. The site of biopsy and the coloring stain used for it, needed to be well documented in the gross description.

Interference of coloring inks in the gross and microscopic interpretation

Surgical coloring inks from Histolab/acrylic paint from Fevicryl acrylic hobby color groups (no. 6, 12, 22, 33) used were excellent inks to color the tissues. The coloring inks helped in tissue identification all through the processing, microscopic examination and forever. We have observed that the inks after drying didn't fade even after several changes in processing solutions. We also found that these inks have never interfered with the stains used for diagnosis, e.g., H and E, histochemical stains, enzyme histochemistry and immunohistochemistry.

We also noted that the agar remained with the tissue all through unlike paraffin. As the agar imbibed minimal/negligible amount of hematoxylin during staining, it remained minimally stained giving a very faint background hue [[Figure 2]a] which did not interfere with H and E stain [[Figure 2]a], histochemical stains [[Figure 2]b, AB-PAS], enzyme histochemistry [[Figure 2]c] or immunohistochemistry [[Figure 2]d] and also during photography.

   Discussion Top

The bacteriological agar that is composed of sub units of galactose is extracted from the cell wall of red algae. [6] Agar is best used for tissue embedding because of its property of hysteresis [6],[7] that is, it remains solid at 36°C ± 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°C ± 1.5°C, a temperature range, which a histokinette never reaches. Thus, the agar remains solid all through the tissue processing and the embedded oriented tissue fragment in agar remains secured and oriented all through the process.

While differentially coloring tissue grossly and embedding one has to make sure that the inked tissue is dry before it is embedded, lest, the ink spreads into the embedding medium. Agar must be poured carefully onto the tissue after it is placed inside the holes in agar. Additionally, the agar must be neither not too cool (jelly like, does not pour) nor too hot (tissue floats and loses its orientation). If the agar fails to enter into the crevices around the tissue, it is insecure, tends to fall out or entrap air pockets. Avoiding air pockets is crucial to hold the tissue securely during further processing and section cutting. One can also use agar-gelatin mixture to make the preembedding matrix stronger. [7] Once the sections were cut and rehydrated 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.

Another advantage was that, during processing, the agar does not hinder in formalin penetrating into fresh tissues embedded in it. [6] Formalin penetrates the agar completely to fix the tissue without disturbing the agar. Thus, this technique of agar-paraffin embedding facilitates tissue fixation, processing and optimal orientation and avoids tissue loss. This technique may also be ideal for embedding precious tru-cut-biopsies from various organs obtained under image guidance.

To carry out this agar embedding technique on a larger scale, we agree with Ventura et al. [5] who have devised a method where in the agar solution could be prepared in large volume, divided as aliquots of 3-5 ml into test tubes and refrigerated. The required number of test tubes could be taken out as required, as per the number of biopsies to be embedded in that sitting and agar is melted either by using a simple water bath or a micro wave at 300 watts for 30 s or 600 watts at 10 s. Setting of the agar would take approximately 8-10 min and the agar blocks can be prepared and kept ready just before the fresh tissue embedding. This is probably an additional step to the routinely practiced tissue embedding technique. Care needs to be taken to see that the agar blocks are prepared fresh and give no scope to become like a culture medium.

We agree that the principle and technique described here for embedding multiple tissues in agar-paraffin block can also be used to prepare a tissue microarray block. [5],[8] We have embedded upto four tissue samples in a block. A single section may feature upto four tissue samples from four different sites from a patient and these can be used for special techniques such as immunohistochemistry provided the origin of tissue and coloring stain used is documented. Occasionally, one could embed >4 biopsies, provided they are colored by four different coloring inks and tissue size is small enough to be accommodated in one block. This improvised technique is advisable for precious tru-cut-biopsies obtained by computed tomography and ultrasonography, small skin, ophthalmic and temporal artery specimens. [9] The traditional coloring ink in use is India ink but when different surface/margin/tissue need to be colored, the other permanent color inks from Histolabs or acrylic paint from Fevicryl acrylic hobby color groups (no. 6, 12, 22, 33) [4] may be employed. These inks stain both fresh and formalin fixed tissues, survive the overnight processing and the subsequent staining procedures on cut sections and are seen clearly in the paraffin blocks as well as microscopically on the periphery of the sections. They do not unduly color the processing fluid or spread within the tissue sections. Unlike inks from Histolabs, these acrylic hobby colors are easily available in most stationary shops, have long shelf life, are non-toxic and can be bought as single units at economical price. [4] Red and magenta inks are preferably avoided as they may get obscured by the eosin of H and E stain at microscopy level.

What was started for rectal biopsies in the evaluation of patients for HD, this improvised double embedding technique was extended for mucosal biopsies other than colon such as corneal, conjunctival, buccal mucosal, vocal cord and endobronchial biopsies and the results obtained were excellent.

   Conclusion Top

The modified agar-paraffin embedding technique represents a simple, reliable, user-friendly method that can greatly improve the quality of diagnostic information one can obtain from minute biopsies by improving on tissue orientation, quality of sections and shortened TAT. It also economizes on number of paraffin blocks, manpower, chemical reagents and laboratory infrastructure.

   Acknowledgement Top

The authors thank Research Society, St. John's Medical College, Bangalore; for supporting this research study; Dr. Divya and Dr. Suravi Mohanty for reviewing the slides and giving their critical comments; all technical staff for assistance in the preparation of tissue sections from agar-paraffin blocks; Department of Pediatric Surgery for Clinical Material and Dr. Kanishka Das for his valuable comments and for reviewing the manuscript.

Prior publication: The paper was presented at the Paediatric Pathology Society (PPS) meeting held at Birmingham, UK on 4-6 September, 2014.

   References Top

Morson BC, Dawson IM. Reception and examination of biopsy and surgical specimen. Gastrointestinal Pathology. 4 th ed. Oxford, London, Massachusetts and Victoria: Blackwell; 2003. p. 3-7.  Back to cited text no. 1
Arnolds WJ. Oriented embedding of small objects in agar-paraffin, with reference marks for serial section reconstruction. Stain Technol 1978;53:287-8.  Back to cited text no. 2
Blewitt ES, Pogmore T, Talbot IC. Double embedding in agar/paraffin wax as an aid to orientation of mucosal biopsies. J Clin Pathol 1982;35:365.  Back to cited text no. 3
Tampi C. In search of the rainbow: Colored inks in surgical pathology. Indian J Pathol Microbiol 2012;55:154-7.  Back to cited text no. 4
[PUBMED]  Medknow Journal  
Ventura L, Bologna M, Ventura T, Colimberti P, Leocata P. Agar specimen orientation technique revisited: A simple and effective method in histopathology. Ann Diagn Pathol 2001;5:107-9.  Back to cited text no. 5
Armise´n R. Agar and agarose biotechnological applications. Hydrobiologia 1991;221:157-66.  Back to cited text no. 6
Jones MV, Calabresi PA. Agar-gelatin for embedding tissues prior to paraffin processing. Biotechniques 2007;42:569-70.  Back to cited text no. 7
Yan P, Seelentag W, Bachmann A, Bosman FT. An agarose matrix facilitates sectioning of tissue microarray blocks. J Histochem Cytochem 2007;55:21-4.  Back to cited text no. 8
LoRusso FJ, Font RL. Use of agar in ophthalmic pathology: A technique to improve the handling and diagnosis of temporal artery biopsies, subfoveal membranes, lens capsules, and other ocular tissues. Ophthalmology 1999;106:2106-8.  Back to cited text no. 9

Correspondence Address:
Dr. Usha Kini
Department of Pathology, St. John's Medical College, St. John's National Academy of Health Sciences, Bengaluru - 560 034, Karnataka
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Source of Support: None, Conflict of Interest: None

DOI: 10.4103/0377-4929.151156

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