Note: The vast majority of surgical pathology labs do not use agar techniques; and, as with so much of routine surgical pathology, agar techniques are not even addressed by, much less approved by the FDA. Agar utilization is a common sense and intuitive trick which can be employed into techniques for the betterment of patient care. However, some of the ways we use agar are in efforts toward exactitude and thoroughness, the degree of which is hotly debated in pathology! [disclaimer]

The American Journal of Dermatopathology
Volume 5 Number 2
April 1983                       
TIPS FOR AND MOSTLY BY TECHNICIANS:
note 2004 update at end of page
Florence Nygaard and Gusstave Mondragon, Editors

CORRECT ORIENTATION OFSPECIMENS FOR HISOLOGIC PROCESSING:
Preliminary embedding in agar.
Ervin B. Shaw, M. D.
Johnnie M. Johnson, HT (ASCP)
Connie G. Watson, HT (ASCP)

Cell-rich body fluids and exudates, fragments of tissue, biopsied specimen, and larger surgical specimens of tissues are routinely prepared for microscopic examination through several procedures: fixation, dehydration, clearing, infiltration with a supportive hardening compound such as paraffin wax, embedment in the hardening material to form blocks, sectioning on the microtome, mounting of sections on glass slides, and staining with various dyes for reading of pathology. All of this comes to a series of options. At a time in the course of disease, the patient presents himself to a physician; the physician selects the site that seems best to him for biopsy (or more major surgery); the pathologist selects from the surgical specimen portions most likely to yield meaningful diagnostic information; and the histotechnologist, after his processing of the specimen secandam artem, selects the best mircotome sections to mount on microscopic slides for interpretation. As the pathologist inspects a tissue sample grossly, he may desire a particular orientation of it within the block.

Proper orientation is most necessary in cases of biopsies of skin in which all structures and layers must be correctly seen, even in common instances of very small samples. This desideratum has in the past been difficult to ensure for many reasons. Markings with India ink or other stains applied during gross examination may wash off during the passage of the samples through the various processing solutions; landmarks noted during gross examination may no longer be discernible enough for proper orientation after mechanical processing because of the action of the chemicals, which may distort the shape of the specimen; and, finally, pieces of tissue may shift around within the processing cassette. Incorrect embedding may then result in tangential sectioning, which may interfere with reading or cause incorrect critical measurements, such as the thickness of a malignant melanoma. There is a previously reported [first by Dr. Lund in 1961] but little-used technique for handling of specimens with which these problems can be overcome.^(1-4)

METHOD

[see our modern method agar recipe] Commercial laboratory-grade dry agar is prepared as a 3% (w/v) aqueous solution in the same way gourmet cooks prepare gravy. Dry agar is added to a small amount of cool tap water sufficient to wet the particles thoroughly. A sufficiently large amount of tap water is brought to a rapid boil, and the wetted agar is added bit by bit to the boiling water while stirring (a magnetic stirring bar may be used). The stirring is kept up for a short time until the solution appears to be clarified, to a point when no particulate matter is visible. The solution is then poured into test tubes for immediate use or for cooling, solidification, and storage. Large batches of tubes may be prepared and maintained in a laboratory refrigerator. Thereafter, as needed sufficient quantities are reliquefied by placing tubes in boiling water, or in a microwave oven, or by holding the stoppered tubes in a 60^oC heat block at the cutting station. In the latter case, one must be certain that the test tubes are filled only to the height of the heat block wells.

By pouring directly from a tube or by dropper pipettes, one can apply an agar puddle to a correctly oriented specimen, allow the agar to cool and solidify, and then process this "preliminary embedded" specimen in the usual way. Several aims are thus achieved, namely:

1. The pathologist is assured of maintenance of desired orientation of specimens from the gut, skin, margins of malignant neoplasms, and thin-walled collapsible cysts.

2. Reduction of "crush artifacts" of delicate tissues (neoplasms, lymph nodes, liver) from excessive pressure by forceps or heavy tamping during final embedment in paraffin molds.

3. Reduction of material costs and working time of personnel by embedding of several specimens from a given case in a single block in a coded arrangement.

4. Aggregation of small pieces of tissue into a single block (bone marrow fragments, endocervical curettings, cutaneous curettings, cell block) for easy final embedding with consequent reduction of risk of "floaters" showing up on slides of other cases.

5. Suspensions of microorganisms in coded blocks to serve as controls of stains.

This preliminary embedding technique works best if attention is given to a number of easily learned technical factors, namely:

1. Use of a flat, cool work surface such as a metal plate or the metal edge of a cutting station sink for arrangement of a specimen.

2. Making the agar puddle block at least 3 mm thick with well-rounded borders to prevent its "popping out" from the microtome block.

3. Beveling by scalpel of the edges of the agar block also helps prevent "popping out."

4. Allowing the agar block, at first clear, to harden to slight opacity before placing the block into the processing cassette. This helps to prevent rounding of desirable flatness of the bottom [down] surface during processing.

5. Seeing to it that agar blocks are not so thick that they do not dehydrate evenly during processing and thereby cause an irregular rather than flat bottom surface to be formed.

6. Taking care that bubbles do not form during pouring or pipetting in the agar block because bubbles tend to retain clearing agents and interfere with the smoothness of sections on slides.

7. Blotting, if necessary, of fixative, mucus, or other sources of moisture before covering with the agar solution.

8. Permitting the agar to solidify completely because fresh tissue is otherwise more difficult than is fixed tissue to hold firmly in a block.

9. Remembering that agar blocks resist drying in room air for about half an hour and therefore batches [of "grossed" cases] must be collected and placed in processing cassettes at proper intervals.

10. Slipping and lifting agar blocks from the work surface by sliding a scalpel blade under them and sliding them off the blade into processing cassettes.

11. Remembering that thin specimens standing on edge tend to flop over when poured agar touches them, and avoiding the error by placing a very small starter drop at edges of specimens.

12. Use of slices ["walls"] of solid agar to retain thin curettings or other fragments difficult to orient in perpendicular positions while melted solutions of agar are applied.

13. Making markers of agar colored black by India ink [or other colors] that may be sliced and show up well on finished slides when used as dividers, or shaped to code margins in tissue blocks.

The same technique is useful in processing some types of frozen sections. It is especially useful in processing frozen sections taken in Mohs surgery in which both proper orientation and marking of margins are critical. Such use of agar is very helpful in handling small specimens that ordinarily would be difficult to place at particular planes for frozen sectioning. Proper orientation of fresh tissue for freezing in order to do immunohistological and enzyme studies is also feasible. Agar freezes on dry ice and thaws without distortion. In cryologic applications, as much excess agar as possible should be trimmed away so as to avoid thick differentials of texture between the solidified embedding compound, ice-hard agar, and frozen tissue.

References:

1. Boldorac, R.: Formalin-agar for embedding small tissue specimens. Lab Med 10: 766, 1979
2. Lund, H.Z., Baker, L.L., and Anthony, J.: Preliminary embedding of small biopsies in Agar: A summary of 15 years experience with the technic. Arch Dermatol 111: 1663-1664, 1975
3. Lund, H.Z., Forrest, W.W., Harris, J.R., and Brown, E.H.: Preliminary embedding in agar-agar. AM J Clin Pathol 36: 562-564, 1961
4. Shackelford, R.I., and Jones, J.L.: An embedding medium for permanent sections of exudative material and fragments of tissue removed for biopsy. Am J Clin Pathol 32: 397-398, 1959.

Write for reprints to: Ervin B. Shaw, M. D. The Lexington County Hospital, 2720 Sunset Boulevard, West Columbia, South Carolina 29169. [none available as of 25 Nov. 2001, so posted on-line]

1. Using an absorbent paper/cloth towel, blot fixative away from tissues before applying agar to make the agar-tissue button (a "watery" button is weak)
2. Once remelted for use, non-fresh agar (in heat block more than a few days) tends to molecularly degrade (I think) so that the dehydration phase of tissue/agar processing will sometimes result in collapse of the agar button into a plastic-like wafer which is hard to microtome. So, we discard any unused liquid agar at the end of each workday.
3. Foreign fibers from such as the prostate "blot papers" will stay attached to the core fragments and interfere with optimal microtoming

[more agar tips] We've continually used agar pre-embedding, in large volume, since 1977; and I would never practice pathology without it!

(posted 9 February 2002; latest update 17 January 2005)