Hundreds of millions of blocks are being produced in histology laboratories all around the world every year. It would not be an overstatement that embedding, sometimes called blocking or casting, is the very important step in histology processing technology that ought to be assigned to the most experienced histotechnologists.
Embedding stands with one leg at the end of grossing and with the other one at the beginning of sectioning with tissue processing in between. Principles of embedding include following grossing instruction and facilitating microtomy in the most efficient way. Embedding technique is a well established procedure that has not been changed for decades, except the speed of performance and the attention to every single item. The first is higher, the second is lower. The seeming simplicity of the procedure is one of the reasons that embedding occupies minimal space in histology technique textbooks and manuals, predominately from the microtomy approach (1, 2, 3, 4).
Embedding automation is knocking on the door of histology practice trying to make manual embedding obsolete. However, there are some methodological and technological locks to open before automatic embedding becomes diagnostically reliable and the cost effective working reality in surgical pathology.
This article tries to discuss embedding automation on the background of an approach to embedding, both, as an extension of grossing orientation and a simple, but skilful technical procedure. The presented materials are concentrated only on the methodological part leaving out technological details of microtomy automatically embedded blocks as well as organizational and economical issues. Although the latter is obviously significant for new technology implementation, this subject requires special statistical study of productivity.
The details of placing the specimen in the sectionable cassette are presented to show some methodological problems of embedding automation. They seem the most important at this stage. Pathologists ought to be aware of these details for the sake of informed judgment on embedding automation feasibility in surgical pathology practice. The goal is to show embedding automation in its development, as work in progress. This subject is not explored in current literature.
Grossing with the eye on the embedding mold
Of course, the grossing sample for embedding should fit the processing cassette, as well as it should not exceed the thickness designated to the processing program. However, the main technical task is that the grossing person should envision the sample’s embedding to assure that the most informative part appears on the slide.
There are many means for special embedding instructions. These include, but are not limited to placing notes in the grossing log, marks on or in the cassette, place some notches, and inking. Some institutions specifically notify the embedding person about tubular organs and arteries, or in special embedding Standard Operating Procedures (SOP) manual’s protocols, for instance in a case of alopecia.
Ink and knife (blade) are the main means for nonverbal communication between the grossing and embedding persons (5). The way the section has been cut gives a clue on how to embed with the lesion or area of interest on the front of the section. In the same way an inked zone should be at the periphery of the embedded specimen/section. In Mohs surgery the interactions between sectioning and inking appears in the most apparent way.
Embedding orientation of biopsies and small specimens has generated a variety of techniques that is evidence that surgical pathology practitioners take orientation seriously (6). There is no any universal orientation method. In the end of the day, however, the quality of embedding always depends on the performance at the embedding station.
Technical details of manual embedding
Let’s explore the technical aspects of manual embedding under the angle of automation feasibility in practical surgical pathology. These details are simple but crucial for professional embedding.
With some hesitation over triviality of description, it is necessary to define the elementary simple technical embedding procedures: the sample is placed with the surface to be cutfacing down at the bottom of the mold with dispensed paraffin according to the instruction, if any. The artisan art includes the race against hardening wax and cooling instruments, although their warming and change is a part of the procedure. Embedding is not a simple task keeping in mind unevenness of the sample’s surfaces, and, of course, multiple fragments with sometimes different consistency. Sometimes a gentle pressure over the specimen is enough to keep embedding flat, but often it is not enough. There are some gadgets which make the specimen flat. For example, now rarely used, aluminum tissue press, called “tamper”, can flatten large surfaces. However, in most cases, especially with multiple fragments, this task is carried out by forceps. Anyway, the sample does not fall as a stone at the bottom of the mold, but is managed by the embedding person. Uneven embedding surface inevitably would require trimming and loss of precious diagnostic material or diagnostically informative part of the specimen can be far away from the microtomy surface. Trivial for professionals in the field, these descriptions have been made just to underline the difficulties that embedding automation has to overcome.
Can automation completely substitute fine and precise operations during manual embedding procedure?
Histology processing without manual embedding
There is a fascinating trend to make manual embedding obsolete by introducing grossing devices that are used as cutting blocks. The cost efficiency of this improvement might be substantial if it were used in large laboratories. The embedding “floaters” would be excluded. The most importantly, the specimen orientation would be carried out during grossing as final. Among the participants of the processing, the grossing person has the most information about the specimen before the slide reaches the pathologist. A completely different methodology in the histology laboratory would emerge.
In the patent application filed in 1996 and 1998 and issued in 2007, Warren Williamson IV et al. claimed the basic technological idea of the change to embedding automation in histology processing (7). The patent is now in public domain and it is legitimately quotable.
The patent’s abstract proposed a fluoropolymer sectionable cassette, or in original description “tissue trapping platform” where “a tissue biopsy sample is placed on a tissue trapping supporting material that can withstand tissue preparation procedures, and which can be cut with microtome”. The sectionable cassette, actually an insert, is placed into a specially designed plastic frame.
Sakura Finetek U.S.A., Inc. offers Tissue-Tek® AutoTEC® Automated Embedding System with Tissue-Tek® Paraform® Sectionable Cassette System. The latter will be the subject of this technical note because AutoTEC® is, by essence, a tissue embedder (developed by Ward Engineering, Inc. Columbus, Ohio) with some robotic elements to cast blocks. The design of the sectionable cassette, however, is crucial for diagnostic value of the slide under the pathologist’s microscope. The goal is to show the sectionable cassette in its development, including the latest variants that Sakura Finetek USA presented in 2009.
There are four main types of the Tissue-Tek Paraform® sectionable cassettes: for large sections (Fig. 1), for middle size sections (Fig. 2), for assorted biopsies (Fig 3) and small biopsies (Fig. 4). Actually, the difference is in the design of Paraform® inserts depending n the type of the specimen. The proprietary resin material used for the inserts is called Paraform®. The inserts are placed in the specially designed plastic frame (Fig. 5) that imitates a regular plastic cassette that is affixed in microtome’s the cassette holder (Fig. 6).
Figure 1. The cassette for large tissue samples.
Figure 2. The cassette for middle size tissue samples.
The difference between these inserts is in size and perforation at the bottom as well as the lid. The latter seals completely the tissue sample in the insert to prevent any movement outside during processing.
In a sectionable cassette of a different design (Figure 3), a small fragment can be locked in. The cassette has a spongy lid and small perforated bottom which was called by the Williamson’s patent a sectionable filter.
Figure 3. The inner cassette for small specimens
More serious task is when the specimen needs orientation or placement for sectioning on the edge. The sectionable cassette (Figure 4), predominately for biopsies, has multiple “pegs”, or “posts” as is described in the Williamson’s patent. The amount of posts can vary from 20 to 40. They can be placed indiscriminately or in some row order. In general, the posts were intended to place a specimen between them to accommodate different configurations, for example of gallbladder section.
Figure 4. Tissue-Tek® sectionable cassette for specimen orientation.
The position of the specimen is secured additionally by the lid that is closed by the grossing person. The insert, or an inner cassette, is placed in an outer frame (Fig. 5) for processing in AutoTec® that generates a block for microtomy. In the AutoTec® , a tamper like device using a robot stages the insert into the base mold by pushing down on the four corners of the insert. Paraffin than is poured, and the block is chilled. The block is compatible with the microtome chuck (Fig. 6).
Figure 5. The plastic outer frame with the Paraform® insert (inner cassette).
Figure 6. The paraffin block ready for microtomy.
A different variant of Tissue-Tek® Paraform® cassettes for biopsies was demonstrated at 35 NSH Convention Trade Show in 2009. They were designated for shave biopsies and for core biopsies (Fig. 7). The design includes four rows of wells with cone like plastic bristles extending from the surface down almost to the bottom of the well. The purpose of the bristles is in immobilization of the specimen at the microtome cutting surface. The difference between them is only in the space between the bristles. It is twice as big in the core biopsy cassette than in the shave cassette. Both types of cassettes have an identical lid with pins.
Figure. 7. The new version of Tissue-Tek® Paraform® cassettes for shave and core biopsies.
A biopsy in the Tissue-Tek® Paraform® Cassette
The positioning of large or middle size tissue sections in the sectionable cassette is a simple and clear act of placing the tissue sample in a diagnostically informative way although some section which require on edge cut need appropriate immobilization. This can be achieved by combination of the thickness/size of the section and immobilized by the insert’s lid. Technical problems of “sectionabily” of the block can be solved by appropriate combination of type of paraffin, durability of the microtome’s blade, and microtomy skills to get a satisfactory microtomy ribbon.
The methodological problems for biopsies and small specimens are more difficult. The main problem is effective immobilization. The prostate core biopsy is taken as an example how the biopsy specimen “behaves” in the Tissue-Tek® Paraform® cassette.
Let’s take a closer look how a biopsy is positioned in the in the inserts. Figure 8 presents an inked prostate needle biopsy core in the sectionable cassette. The core definitely has space for horizontal and vertical movements during conventional tissue processing or microwave assisted processing.
Figure 8. An inked prostate needle biopsy core in the Tissue-Tek® Paraform® sectionable cassette.
Of course, a tubular organ, tube or artery also might not remain in original position falling down or tilt leading to wrong section. An insignificant tilt of a skin section can cause misleading tangent microsections which might have diagnostic significance.
Even thicker breast core biopsy fragments (needle biopsies often have fragments of different size and configuration) do not occupy the entire space between posts.
Figure 9. Fragments of breast needle biopsy in Tissue-Tek® Paraform® cassette.
These two examples show that biopsy specimens are positioned uncertainly to get complete sections of the most representative material. Experimentation with biopsies confirmed that core and small fragments drifted from original position during conventional processing. It is a well known fact the in a case of breast biopsy one can be able to find on a single slide two or more different pathologies, and the pathologist cannot afford to loose anything due to technical problems.
The immobilization of the biopsy specimen is the main embedding automation sticking point. The designers of the sectionable cassette definitely wanted to overcome this obstacle. The 2009 new version of Tissue-Tek® Paraform® cassettes try to provide more efficient immobilization .
As it can be seen on Fig. 10, the prostate core is at the bottom of the cassette additionally immobilized by cone shaped bristles. This is a definite step ahead in immobilization. However, if the cores were not flat nothing is able to do this. The picture shows that the bent part can be cut off by the trimming until the blade reaches the main core or requires numerous sections.
Figure 10. A new version of Tissue-Tek® Paraform® cassette with a bent prostate core biopsy.
A different problem occurs, if cores are different length or uneven. The Fig. 11 shows this situation. How to make flat the core if the bristles prevent from putting an instrument for this purpose? The small fragment can be lost through the perforations on the side of the cassette because it is not held by the cone bristles.
Figure 11. A new version of the Tissue-Tek® Paraform® with two uneven prostate biopsy cores.
As it is well know, even prostate core biopsies are different in thickness, never mind breast or kidney core biopsies. The Fig. 12 presents a situation when the core remains on the bristles and requires some effort to put it on the bottom of the cassette. By the way, as it occurred in this case some fragments fell apart and required additional cassette because if left alone they would escape from the cassette during conventional processing. The problem of very small fragments will be discussed later.
Figure 12. A fragmented prostate biopsy core in the new version of Tissue-Tek® Paraform® cassette.
One of the features the new version of the Tissue-Tek® Paraform® cassette is the redesigned lid with four rows of pins (Fig. 13).
Figure 13. The pins at the lid of the new version of the Tissue-Tek® Paraform® cassette.
Unfortunately, they are too short to make a difference in flattening the thin core on the bottom of the cassette and too firm by piercing fragile and diagnostically important areas of thick cores, for example in breast biopsies.
Even in the case of a shave biopsy these pins are not only useless but in some occasions they might be damaging for flattening the surface of the section. It is unclear how the pins will interact with the large shave biopsy fragments or sections. Perhaps, they should be eliminated because the bristles can hold secure the inserted shave biopsy or section of it, except very small and thin.
Of course, small biopsies or small fragments of biopsies require special treatment. The cassette for small biopsy specimens, in general, can be acceptable (Fig. 3). However, the spongy lid is not the best option. As the Fig. 14 presents, the small fragment of a core biopsy is comparable with holes in the lattice wire of the sponge. The fragment can be trapped in this net under conditions of vacuum and pressure processing. There are some literature data of the damaging effect of polyester foam on fragile biopsy material (6). And by the way, filtration of uncountable mucinous material is difficult to carry out technically at the small space of the cassette. How to aim amorphous material in such a small as 0.7mm diameter space? This detail is substantial in everyday surgical pathology grossing practice.
Figure 14. A fragment of a prostate core biopsy in the Tissue-Tek® Paraform® cassette for small biopsies.
The example of prostate core biopsy’s options in the embedding automation cassette might show the obstacles that this method faces and how gradually they are solved. The TissueTek®Paraform® cassette needs further “evolution”.
During 34th NSH Convention at the Trade Show in September 2008, QuikMbedTM silicone sectionable pad, QuickMBed, LLC, Bethesda, MD) were demonstrated for automatic embedding.QuickMBed, LLC distributed only for demonstration and testing a blue silicone pad that has a flexible base and rows of bowling pins like stems with flared ends on the top of the stems. The pad can be placed in a standard plastic cassette. The sectionable pad is intended for needle core biopsies and shaves skin biopsies according to the QuickMBed website (www. quickmbed.com).
Figure 15. QuickMBedTM silicone pad
The Figure 16 shows a prostate needle biopsy core between stems of a flexible silicone pad. The grasp of the stems is completely secure. Even if the pad falls on the floor, the specimen remains at the same place. It looks that the QuickMBedTM flexible silicone pad is very useful for cores and thin specimens.
Figure 16. A prostate needle biopsy core between stems of QuickMBedTM pad
Unfortunately, QuickMBed abandoned the silicone pad due to legal disputes of patent infringement with Sakura Finetek USA. Even the domain QuickMBed. com has been deleted.
In 2009, QUICKMBED, Inc. filed an international patent application “Scaffold for tissue sample orientation” which comprises hyrdrogel or organogel. It is flexible to permit to be bent, maintains a tissue sample in a particular pre-determined orientation, and not microtome sectionable. The abstract of the patent application states that the substantially liquid component can be replaced during fixation and processing with components that are substantially solid, making the scaffold microtome sectionable. A scaffold is not enough for introducing embedding automation. Immobilization of the specimen in the platform ought to be provided. Otherwise the sectionable scaffold is meaningless.
Preliminary results of embedding automation with plans for clinical studies “to quantify the efficiencies achieved by Sakura Finetek’s paraform system” have been published in 1999 (8). There is no any study of automated embedding in the available literature. In article “Automated histology- many labs, many test” in CAP TODAY magazine, automated embedding was mentioned among other equipment for automation in histology practice (9).
There are many embedding automation technical issues important for histology laboratory as “sectionability” of the paraffin block that contains additional to wax substances, interaction with microtome blade, particularities of trimming, adhesion to the glass slide, and relationship with staining procedures. However, immobilization of the grossing orientation is the main methodological challenge that makes all mentioned above important issues irrelevant if the specimen immobilization problem were not resolved. The completeness of biopsy submission can be jeopardized. The surface presented for microtomy should be even and exactly as to be intended at the end of grossing. As a person who spent considerable time at both sides of the microscope (reading slides and grossing), the author can responsibly state the grim truth that the pathologists are unable in many occasions to control embedding outcomes, completeness and adequacy of the material on the slides.
Autopsy and large specimens are the first in the line for embedding automation. There is always space for grossing and sectioning manipulations. A slight unevenness of the section can be straightened out by trimming.
Biopsies are completely different animal as far as correct orientation is concerned. Everyone who does grossing biopsies on a regular basis knows that even prostate core biopsies taken by the same clinician with the same needle under the same conditions in many cases are different in length, thickness, even consistency. Although, for example skin shave biopsy can be definitely positioned for manual embedding orientation, it can not be securely lined up in all instances for automation embedding. In this situation a different criteria of specimen immobilization should be applied.
The inventors of sectionable cassette clearly understood snags in the way of embedding orientation. In the Williamson’s at al. patent description there is a chapter “Immobilization of tissue on Platform(7)” Different options of adhesives were suggested “…to keep the tissue samples in place while in fluid medium of the issue processor”. For example, cyanoacrilate was recommended to bond larger tissue samples to the non sectionable stage. A variety of adhesives were suggested for biopsies from sophisticated ballistic particle deposition coated with protein to spraying a thin glue-like substance, agar or other gel. The variety of proposals for immobilization of the specimen in the cassette reflects that the inventors of embedding automation took this issue seriously living the concrete technological solution for further development of the method.
The Williamson’s patent has some provisions for immobilization in the cassette design. Among them, there is a proposal of a biopsy cassette close to the new version of Tissue-Tek® Paraform® (Fig. 7, 10-13). However, there are substantial important differences. The narrow well for the biopsy specimen in Williamson’s patent is secured by a snap fit locked lid (top element) with multiple projections. The projections are thin to prevent any damage to the specimen and long enough to extend to 1/2 mm from the bottom of the well. The projections work as tissue-retaining feathers which trap the biopsy specimen against the bottom of the well making the biopsy specimen in one plane, e.g. providing gentle effective immobilization.
The example of Williamson’s et al. patent is interesting by itself as a reflection of interactions of science, artisan art of histotechnology and manufacturing realities. The brilliant idea collides with technological opportunities which are limited predominately by economical considerations to make an affordable for regular laboratories product.
Sakura Finetek without any doubt is aware of Williamson’s et al. patent’s the patent proposals. There is, however, a distance between the theoretical idea and its implementation. There are technological difficulties to combine in one piece of polymer stamped cassette a firm frame and a soft lid of it. Anyway, a lid with rigid firm short thorn like plastic pins is not a solution for immobilization of the fragile diversified in size and consistency biopsies.
Williamson’s patent has other features that are still not used. For example, one of the patent’s provisions includes sectionable filters designed to be used to collect biopsy samples from various biopsy containers or devices. These trapping filters can be manufactured in various pore sizes. The suggestion goes far enough to place the specimen, for example by Fine Needle Aspiration or other surgical suction device, directly during biopsy, or as is stated in the patent “at the time of harvest in the surgical setting.”
Embedding automation opens an opportunity for clinician to orient the specimen at the moment of the biopsy. The clinician sees the specimen literally fresh without alterations of natural colors by fixation, when blood is oozing from the place the biopsy that makes orientation easier and more precise. By the way, at the dawn of the endoscopic gastroenterology, clinicians tried to orient some biopsies, duodenal for instance, on cucumber slabs.
For understandable reasons, experimentations with such irreplaceable material as biopsies are limited (by the way, all biopsy materials presented in this study was eventually processed by using conventional manual embedding). Perhaps, it would be reasonable to conduct an experimental study using autopsy and animal material. This study can go in parallel with clinical testing if the latter is conducted cautiously with awareness of possible contraindications for use embedding automation, especially in biopsies.
Meanwhile, the surgical pathology practice might take advantage of the excellent opportunity of grossing orientation for manual embedding that Sakura Finetek’s paraform cassette can offer. It provides a simple and reliable grasp of the specimen or section keeping it as an instruction for orientation during manual embedding. This author published a special technical note about embedding orientation by an incision in a ubiquitous blue foam pad for skin tips, shave biopsies, and some gynecology or gastrointestinal biopsies(5). The mentioned Sakura Finetek’s cassette is a different option. There are definite advantages of using Sakura Finetek sectionable cassette with prongs. The Paraform® inserts with or without the lid can be placed in regular plastic cassettes. They can be successfully employed during grossing for specimen orientation in manual embedding (10). The same can be stated regarding the QuickMBed pad.
While employing sectionable cassettes for manual embedding, the histology practice will become used to this type of cassettes. During manual orientation might be found more appropriate designs of cassettes for embedding automation. In author view, there would be a variety of sectionable cassettes customized to different types of specimens, definitely more than four types currently presented by Sakura Finetek.
If an idea becomes technologically feasible, it will be implemented in practice. Eventually embedding automation will replace manual very much artisan procedure. Surgical pathology community has to be prepared for this shift. Implementation of revolutionary for histotechnology embedding automation with a variety of cassettes requires a different working space and a redesign of the standard grossing tables. Commercial laboratories, probably, will be the first institutions interested and capable to implement the embedding automation. They have the advantage of “monoculture” production line with predominately dermatopathology biopsy specimens.
There are other opportunities that embedding automation is opening. Among them are envisioned in the Williamson’s patent digital gross pathology which is now developed by MILESTONE Medical and cassette bar coding that is not any novelty anymore.
This author definitely believe that the grossing person has exclusive knowledge, means and responsibility for correct embedding of the specimen and its ultimate appearance on the slide for a optimal diagnostic outcome of the case. The grossing person ought to be the last person before processing and microtomy. This is the main reason of advocating and propagating embedding automation.
Can embedding be automated? The answer is “Yes”. More, embedding automation is inevitable. For autopsies and large specimens, there is no conceptual methodological objection for implementation of sectionable cassette. However, biopsies require a technological advance. There is a necessity to solve main problem during processing as immobilization of the specimen after grossing and alignment of the microtomy surface to provide complete and representative material on the slide. It looks as optimal a combination of adhesive/s and special design of specimen holding platform of the sectionable cassette. Gradually, an optimal mode will be found by broader experimentations and implementation in every day practice.
For the immediate future, it is essential to conduct the constructive debate with respect for its complexity and stark choices. This technical note can be treated as a step on the long way ahead.
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3.CarsonFLand Hladik Ch Histotechnology: A Self-Instructional Text.Chicago: ASCP Press, 2009, pp 1-23
4. Bancroft JD, Gamble M: Theory and Practice of Histological Techniques. 5th EdNew York,NY, Churchill -Livingstone, 2002
5. Dimenstein IB: Grossing biopsies: an introduction to general principles and techniques. Annals of Diagnostic Pathology, 2009, Vol. 13, No. 2 pp. 106-113.
6. Dimenstein IB. Incision in Foam Pad for Embedding Orientation. Journal of Histotechnology, 2008 Vol 31, No.3 p. 141-142
7. Williamson, IV, Warren P. et al.: Apparatus and method for harvesting and handling tissue samples for biopsy analysis. United States Patent Full-Text Image database: 7156814. Available at:http://www.patentstorm.us/patents/7156814/description.html. Accessed October 28, 2009
8. Diederichsen Chris, Whitlatch Stephen. Description and Preliminary Results of a Novel Cassette system (Tissue-tek Paraform Cassette System). HistoLogic 1999 31(2), pp. 28-30
9. Karen Lusky: Automation histology- many labs, many tales. CAP TODAY October 2007
10. Dimenstein IB. New Devices for Manual Embedding Orientation. Journal of Histotechnology 2009, Vol. 32, No. 3, pp.123-125
Disclaimer: The author does not have any business relationships with the manufacturer of devices for embedding automation.
Thanks to Gilles Lefebvre (Sakura Finetek USA) for help in preparation of these materials.