(1) Find the nodes...specimen agar pre-embedding...nodes for mets: We've used agar pre-embedding to great advantage since about 1976 [Am. J. Dermatopathology paper published 1983]....[recipe for agar]. In early October 2002, it became apparent that we needed to routinely intensely process certain malignancy-staging multi-nodal lymph node biopsies (such as with any SLN...our SLN surgeon/nuclear med protocol) or dissections (breast cancer cases, melanoma cases, Merkle cell carcinoma cases)...our "intense lymph node processing protocol":

We had years ago created a methodology (several hours fixation, at least)...very commonly overnight (but avoid more than 48 hours)...in Hartmann's fixative (an alcoholic formalin solution containing some acetic acid) to carefully recover all nodes 1-2mm or larger from any specimen. This fixative turns nodes...nucleus-rich tissue...white (turns DNA-rich tissue white for about 48 hours and then white color starts to fade in some cases). All (unless clearly grossly [naked eye] positive for metastatic tumor...in which instance a single piece of each positive node...to view hilar aspect and any suspected transcapsular invasion...is processed for confirmation) node candidates are totally submitted for microscopic study as node pieces no thicker than 3-4mm, all agar pre-embedded into blocks trimmed to fit standard processing cassettes. One needs to have dissection and detection done with dogged determination by an individual with personality compulsive and determined enough to find them all. As at least one expert pathologist has actually published, we saw in about 2000 that our pathologist assistant had the right temperament. So, since then, we have had node dissections done by either of two pathologist assistants. In the absence of such thoroughness, about 25% of breast cancer cases that actually have positive lymph nodes will not have their nodes exposed and correctly diagnosed as positive. And the case will be mistakenly classified as "node negative" and treatment tactics executed for a node negative situation (though it truly is node positive)...the case is, thusly, a "false negative" work up as to node status. 

(2) Levels of section of nodes: Then we desired to be able to prove that the histotechs actually step-cut at "provable", regularly spaced levels (stepcuts) entirely through (stepcut slides produced at no less frequent intervals than every 1.0 mm) each block. PROBLEM: for fear of cutting tissue entirely away (unexpectedly), histotechs are tentative and intuitive in performing routine classical stepcut sections. At that rate, node dissection cases would represent a massive investment in time and effort...unless there were a way to have step-cut depth markers embedded with the nodes in the paraffin blocks. Once set with a method to be quickly sure of the plane of each spaced level, then microtoming between the levels can be performed with rapidity...almost with reckless abandon. This serves to "back out" a great deal of the time-and-anxiety expense of the classical sectioning approach to node stepcuts. Our experienced histotechs estimate that a routine block sectioned for one H&E takes less than a minute...the "intense node protocol" on one block may be as much as 3 times more time...a trivial increase in time from a time-cost and health-cost standpoint.

We conceived of a process whereby we use three different tissue grossing dyes to make sheets of colored agar [making agar] about 1mm in thickness. [In practice, the thickness of agar & the colored layers varies; so, we try to use a slice of the agar that is about as thick as the thickness of the node pieces in a particular block. The point is to get stepcuts from about an outer third, about a center third, and about the opposite outer third of the node-block tissue thickness. In early 2007, we had a challenging case [L-07-2188] in which we were shocked that the sentinel node was negative. So we amplified our intensity to 4 slides per block: first in first color, second in middle color...also an IHC @ this level, 3rd in the 3rd color and 4th nearly thru that 3rd color.] Firstly, in separate batches, we add one of the dyes to the colorless melted agar...making batches of colored agar kept temporarily liquid in the heat block. One then pours/pipettes a previously determined, measured amount of one color of agar into a plastic Petri dish, lets the first layer "softly harden", pours the second color likewise, and then pours the 3rd color. Then, following cooling to room temp, the multicolored agar is sliced into narrow tri-colored solid agar depth sticks...either ahead of time or as needed. One agar depth stick is placed into each cluster of node cross-sections (always with the same color "down",  showing on block face), and this agar depth stick serves as a microtoming level marker, much as an interstate highway mile marker, each change in color proving increasing depth of sectioning.

One can use green, yellow, and red layers as with a traffic light, the green layer being "up" and the final color zone at the deep (interior) paraffin block aspect. Red = start with care; yellow = "you are now to the midway zone"; and green= "get set to stop after cutting in a few more turns". 

If you also use colored (such as black India-ink-colored) agar dividers (specimen separators) to isolate the groups of 2 or 3 node cross-sections, be sure that they are a color entirely different from the initial color of the depth stick (it is the seeing of that initial depth stick color showing in the parafin-block face that triggers the unmistakable command to the histotech to cut "the intense node protocol").

Large number of blocks:  (1) since different histotechs have their microtomes set slightly differently, one can waste much tissue by mounting and remounting and recutting blocks; so, (2) cut all slides of the intense protocol at one sitting. (3) one can "focus", if desired, and do H&E stains first; then do IHC only on the blocks which are not positive by H&E [LMC-03-637]. That is, always do all cutting at one sitting; save any focused selectivity effort for the staining & "reading" phases.

Small nodes: And, when multiple small nodes are agar pre-embedded in a single block along with an agar depth stick, I highly recommend that one or two small, shaped black agar markers be added as triangulators. Then, if one has microscopic node positivity in less than all of the stepcut levels, it will be easy to match-up the pattern of the colored agar markers and correctly decide the true number of positive nodes [LMC-03-232] (the small-node profiles can change shape dramatically through the stepcut series).

For carcinoma cases, we cut an H&E at the start of each agar color & at the end of the deepest color and an IHC slides at each of the 4 levels and also obtain 2 slides for IHC in the middle zone (one is for negative control because not at all uncommon to have anthracotic, melanotic or tattoo [LMC-07-1019 ] dermatopathic pigment). With pankeratin...AE1/AE3.. we had a pleomorphic lobular case that was pankeratin negative [LMC-05-2494] and have changed to using ck7 in April 2005. Because more than one slide stands as unreimbursible expense, we only routinely IHC the slide near the block center. For melanoma cases, we cut an H&E at the start of each color and also obtain 4 slides for IHC in the middle zone (one for S100, one for pan-melanoma and two for negative controls). [protocol] [possible publication, our positive-impact experience] [predicting melanoma cases likely to have positive nodes]

"Thumbnail" images of an agar depth stick in a block of node cross-sections cut about 3 mm thick. In this instance, I pre-embedded the stick & tissue in clear agar; the block is balanced on thumb-tip and finger for the photograph. The colored agar shows up microscopically as a permanent record on each step-cut slide, the color telling which third of the block thickness the slide came from.

Plastic Petri dish containing fused layers of colored agar...then sliced into "depth sticks".

The tricolored depth stick can be similarly used on 2-5 mm skin punch biopsies for obtaining properly spaced stepcut sections.  [S-04-3043]

By making thinner colored-agar sheets, one might be able to indicate step levels in other types of specimens such as core biopsies or endoscopic biopsies.

(3) Full sections: Also, one could use a thin colored-agar "stick/layer" within a clear agar block to indicate that the histotech "face off" the block until the colored agar disappears...rapidly indicating (without fear of "cutting away" the tissue) that the microtome ribbon should now contain complete tissue sections. 

(4) Agar stools: Though I've occasionally used the "stools" in past years, I started their fairly routine use on FNA03-70 (3/27/03) thin FNA cores, scant cell blocks, endocervical currettage, and possibly for small skin punch biopsies, bisected pigmented skin shaves, and thin biopty-gun cores (prostate, liver, medical kidney, etc.). For some reason, there were problems (the histotechs said that the stool "popped out" of the block)...so, we stopped after a couple months (the "press" of daily grossing would have stopped routine use of stools by 2006, anyhow). The sample is carefully aggregated onto the surface of a small square/rectangle/whatever of clear agar the half the thickness of a glass slide (or one can coat the bottom of a metal embedding "boat" with liquid agar. Then a touch of liquid agar is applied by pipette to the surface to hold the aggregate; & then a puddle of liquid agar onto it all...and the stool & tissue moved slightly so that new agar gets under edges of the "stool". This allows plenty of block surface for "facing off" prior to entering the zone of...and saving...the tissue ribbon and/or making slides for H&E and IHC. Agar stools are good for thin cores or any other specimen likely to lose 25% or more of itself in the facing off of the block. By the way, the new disposable microtome blades cut so sharply that the problem of agar "popping" out of the block is a non-issue.

(5) Agar swirl embedding: a small aggregate of fine tissue pieces (ECC or scant marrow particles recovered from a bone marrow aspirate) is placed together on the metal table top and a few drops of agar pipetted onto them. Then, quickly before agar solidification, use a tissue pick to stir the particles slightly "up" into the agar button. This dissociates the particles but keeps them from being wasted away when facing the block off.

(6) Marking sequence or position: (a) Additionally, colored and/or especially shaped markers can be used to say "start here" when several small, separately labeled specimens are agar pre-embedded in the same block. (b) Colored sticks can be used to separate biopsy "A" from "B" from "C" and/or to separate the pieces of two or more cross-sectioned nodes within a single block. (c) And colored sticks can be used to indicate directional information (the deep/internalward tip of the core touches a colored agar "deep end" marker bar) regarding specimen orientation (especially useful in our prostate biopsy process, linked below).

prostate cores thumbnail;... & additional link to prostate series. And, note reference, below to a 2010 article documenting the intuative advantages of ascertaining that cancer is in the peripheral end (PE) of a core².

(7) An aid in frozen section: one can "tack" serial cross-sections of, say, a small excision of a skin cancer for margins (margins can marked with dyes) together so that one can do a one-block FS instead of a block for each cross-sectional piece. Use just enough agar to keep the pieces in proper arrangement to slide off into the OTC-coated microtome chuck (too much agar interferes with FS microtomy).[LMC-03-2907] I believe that this agar technique (I've used it for over 20 years) is an acceptable alternative to the impressive results of S. R. Peters, M.D. steel-well cryo-mold technique¹. His technique is possibly preferable in Mohs-surgery practices: I saw him demo this technique in San Diego 9/03. But, in July 2005, our Ted Mitchell invented a technique [LMC-05-6389] wherein serial slices are oriented with the edge to be checked placed "down" on a routine glass histology slide & the sample gently covered with OTC "goo"...then the specimen-carrying slide is carefully placed on metal in the cryostat to solidify frozen. In the meantime an OCT spot is frozen to cause a  a flat "up" surface on a frozen-section "chuck". The glass slide carrying the specimen is then inverted over the frozen chuck & "finger heat" delivered by  touching over the specimen location in the slide to loosen sample from the slide as it sticks to the frozen OTC of the chuck. More OCT is added to the chuck to fill in around the specimen spaces.

(8) agar en block resection pre-embedding & processing: the intact fixed and dye-marked specimen is placed whole & submerged in liquid agar...agar hardens & then specimen serially sliced with new-sharp blades or a razor knife & the cross-sectioned tissue remains intact (held together by the solid agar...cross-sections then trimmed of agar and oriented and agar re-embedded. [a spectacular case example, images, & more detail]

(9) skin lesions, avoiding tangential artifact: if skin cross-ections get tilted when embedded, an artifact is produced which makes the epidermis seem thicker (acanthotic) & can make a thin melanoma measure a little thicker than is true. This is avoided by cross-sectioning the excision with a very sharp blade (& avoid compression artifact, thereby) and arranging the cross-sections in series with edges all in flat contact with the embedding surface (or carefully orienting the punch or shave biopsy) so that the epidermis is at exactly 90 degrees to the embedding surface & the position solidified in place with agar.

(10) skin lesions for margins:

• elliptical excisions: as in (9) above, one can excellently view the margins.
• punch excision: these can be embedded epidermal surface top down and agar-securred into position and then step cut serially from epidermal surface to on down into subdermal aspect [S10-10922].

(11) skin for alopecia assessment:

• embed punch longitudinally: rotate punch until one sees a hair & position hair so that it is parallel to the embedding surface, possibly allowing a section to display the full hair length.
• embed punch after serial cross-sections: embed in the exact manner you, as diagnosing pathologist, desire them & in one block.

(12) total view of segmental structure: temporal artery biopsies are cut into short segments, arranged on the grossing table surface, and agar pre-embedded so as to have all segments in a single block for a single slide exam.

References:

1. Peters SR, "The Art of Embedding Tissue for Frozen Section. Part I: A System for Precision Face Down Cry embedding of Tissues Using Freezing-Temperature Embedding Wells", The J. of Histotechnology 26(1):11-28. [see process & photos]
2. Ponholzer A, et. al., "Postate cancer at the peripheral end of a prostate biopsy specimen as assessed by a novel marking technique may indicate increased risk of locally advanced disease", Prostate Cancer and Prostatic Disease, pages 1-5, ePUB 9 November 2010.
3. Rosai J, 9th Edition:Rosai and Ackerman's Surgical Pathology, 2 vol. text 2977 pages, see page 2952, 2004.

(posted 24 November 2002; latest addition 9 February 2012)