Pathology Associates Of Lexington, P.A.
Pathology Associates Of Lexington, P.A.
Pathology Associates Of Lexington, P.A.
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        Agar Depth Sticks, markers, stools, etc.
      

The use of agar for the following purposes is little-known, and use of agar at all is probably only to a limited degree & only in a worldwide minority of patient-focused anatomic pathology labs...most NOT using agar techniques at all.

The URL for this page is: http://www.palpath.com/MedicalTestPages/agards.htm

Our practice began in 1971 at the new Lexington County Hospital (now Lexington Medical center), West Columbia, S. C.; and we have continually used the agar pre-embedding technique to a great degree since 1977. I got the idea from newly local pathologist, Hoke F. "Rick" Henderson, M. D. (still of Columbia, S. C.) who'd gotten it from the H. Z. "Herb" Lund, M. D.'s (URL to a memorial, here) dermatopathlogy lab ("Greensboro Pathology Associates" which Dr. Lund founded in 1954 in Greensboro, N. C.), while he spent Dermatopathology Fellowship time there. Dr. Lund was one of the original 12 founding (1963) members in the American Society of Dermatopathology (ASD) and president of ASD in 1967. Apparently Dr. Lund was using this at least as early as 19609 (publishing it initially in 196110). I view him as the pathologist "father" of agar pre-embedding. Decades later, I got a set of typed presentation or publication notes from 19765. According to Shackelford11, using agar was possibly first reported for use in frozen sections in 19437 and then 195112 & 195413. Shackleford & Jones (two "tissue technologists") were the first11 to report the use of agar pre-embedding for permanent sections in 1959. So, agar pre-embedding for permanent section human histopathology slide production has been the subject of reports since 1959 and especially after endoscopy began to result in small biopsies8-11.

Ink/dye-colored agar: We independently saw uses early on for gara markers with color. Years later, I found that the first report of using agar with ink to color it was in19828. Our so-called "intense protocol" with "agar depth sticks" with 3 layers of different color for staging of lymph nodes in breast & melanioma (and maybe other) node dissection cases may not be currently used in any other lab in the world. The majority of surgical pathology labs worldwide do not completely process apparently "negative" nodes (as of 2000). Node examination techniques and intensity of effort are not even addressed by, much less approved by, the US FDA. Varying efforts at thoroughness and intensity of lymph node examination are a common sense and intuitive level for decisions based on many factors, including the betterment of patient care. However, such varying efforts have been hotly debated for decades as to whether there is any real benefit of increasing thoroughness and intensity of effort to the betterment of patient care. 

 (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, internal link here]....[recipe for 3% w/v agar, internal link here]. 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, internal link here) or dissections (breast cancer cases, melanoma cases, Merkle cell carcinoma cases)...our "intense lymph node processing protocol", internal link here:

In about 1975 (and to this day...Nov. 2016), we created a methodology (several hours fixation, at least)...very commonly overnight (but avoid more than 48 hours)...in Hartmann's fixative...internal link here (an alcoholic formalin solution containing some acetic acid) to carefully recover all nodes 1-2mm or larger from any specimen. We still do this technique (as of Nov. 2016). KEY ADVANTAGE of HARTMANNS: This fixative turns any nucleus-rich tissue...such as lymph nodes...WHITE (turns DNA-rich tissue white for about 48 hours and then the 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 three 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 (agar depth sticks): 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 colored-agar 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 wreckless 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 dyes4 to make sheets of colored agar [how made...internal link here] 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 totally 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". 

Separator bars (identify more than one node in a block): If you also use colored (such as black India-ink-colored [how made, internal link here]) 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, sectioning efficiency:  (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.

Step-cut tracking of 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 internal link here] [possible publication...internal link here...of our positive-impact experience] [predicting melanoma cases likely to have positive nodes...internal link here]

agards1.JPG (363423 bytes) "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.

Petri_dish.jpg (120782 bytes)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 indicator: 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 half the thickness of a glass slide (or one could alternatively coat the interior central 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:
URL = http://www.palpath.com/MedicalTestPages/agards.htm#marrow

  • bone marrow surface area-- In about 1978, we two hospital pathologists (Calvert & Shaw) were still performing on the patient all inpatient bone marrows and doing so mostly with sternal marrows using the Illinois needle. That year, Dr. Shaw started doing an initial short aspirate for slide making followed by tilting and rotating the needle within the sternal cavity about one complete turn followed by a suction aspiration to remove some marrow volume. The aspirate was quickly expressed into 10% NBF for purposes of removing blood and clottable proteins. Back in the lab, the particles we sifted from the formalin with a strainer, gathered onto the metal grossing table, and agar pre-embedded, swirled into melted agar to form a particle block about the diameter of a penny or nickle (8-10 mm). In current times, the particles could be quickly removed, agar preembedded and placed into a mercuric fixative or a zinc-formalin fixative such as GI-Fix by BBC. It should be possible to modify technique with other sites and marrow acquisition techniques to obtain a bulk of particles to make a lot of viewable surface are for routine and special viewing of histology. This method strongly relates to the issue of marrow adequacy (internal link HERE).
  • small particles-- 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. HERE...internal link here [This URL = http://www.palpath.com/MedicalTestPages/agarbmclot.htm]...is a process to maximize bone marrow particle surface for histopathology interpretation.

(6) Marking sequence or position: (a) Additionally, colored [how made...internal link here] 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: also, in placenta exams, when 3 umbilical cord sections...toward baby; cord mid-length; and at placenta, a starter black marker at baby's end & the 3 cross-sections embedded in sequence and agar pre-embedded into one 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).

pp6c.gif (64194 bytes)prostate cores thumbnail;... & additional link to prostate process series...internal link here. 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: I saw mention of this 1979 article6 in the early 1980s. 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 the 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, large-block pre-embedding & slicing & 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...internal link here ]

(9) skin lesions & GI biopsies6, avoiding tangential artifact: if skin cross-sections 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. This is my Letter to the Editor retort about other methods being as good as agar pre-embedding, URL to that letter HERE.

(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.

(13) Contents within a structure11: agar can solidify around endobronchial, intracystic, intra-abscess and other materials to maintain spatial relationships of materials and anatomic structures.

(14) Tissue Cultures5: Ken's was adapted by Pat Alexander, HT(ASCP) using a stainless stell embedding mold. Liquid agar is poured into the mold to solidify. Then a small area is scooped out and sub-totally filled with the tissue culture cellularity. Then liquid agr is paled on top of that area to seal it; and the block is trimmed & re-embedded.

(15) Cytology fluids cell blocks5 & 6: mucous is broken up and concentrated sample is fixed and then reconcentrated (centrifuged, for example). The concentrate has as much supernate fluid removed as reasonable. Then liquid agar is addedslightly stirred to suspend the cellularity. When solid, it can be dislodged and cut to size and agar re-embedded as if small biopsies.

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, 2004. [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. [2011 update HERE]
  3. Rosai J, 9th Edition: Rosai and Ackerman's Surgical Pathology, 2 vol. text 2977 pages, see page 2952, 2004.
  4. Dyes: we wanted dyes that would not fade during processing or slide and block storage; initially we used body tattoo dyes; we now use BBC Path-Mark dyes or maybe Triangle Biomedical Sciences, Inc., Durham, N. C., USA.
  5. Kenneth Alexander, HT(ASCP), August 1976, typed notes, "The Use of Agar in Processing Minute Specimens", either to a National Society for Histotechnology meeting or publication.
  6. Ruby Boldorac, HT(ASCP), Creative Imagination section, "Formalin-Agar for Embedding Small Tissue Specimens, Laboratory Medicine 10(12):766, Dec. 1979, [not found online & text transferred to this website, HERE].
  7. Foley, J. O., "Protargol Method for Staining Nerve Fibers in frozen or Celloidin Sections", Stain Technology 18:27-33, 1943 [HERE]. BUT, viewing (11/12/2016) what I can online, I don't see reference to agar. I do not recall where I obtained this reference.
  8. Bourgeos N., et. al., and Buyssens, "Agar-paraffin Embedding Technique Applied to Small Diagnostic Biopsies", Stain Technology, 57(4):251-254, 1982, PMID 6755805 [mentioned, Letters, Dr. Buyssens of Antwerp, Belgium (HERE); American J. of Dermatopathology 5(5):514, October 1983].
  9. Lund H. Z., et. al., "Preliminary Embedding of Small Biopsies in Agar: A Summary of 15 Years Experience With the Technic", Archives of Dermatology 111:1963-1964, 1975 (now JAMA Dermatology beginning Jan. 2013).
  10. Lund HZ, Forest WW, Harris JR, Brown EH, "Preliminary embedding in agar-agar, Its Value in the Processing of Multiple Separately Identified Specimens, and in the Orientation of Small Biopsy Specimens", Am. J. Clin. Path. 36(6): 562-564, 1961 [does not show on-line except that Google Scholar directs to Technical Bulletin of the Registry of Medical Technologists, 31:192-194, 1961, (HERE) which does not show the text, Nov. 2016].
  11. Shackelford RI and Jones JL, "An Embedding Medium for Permanet Sections of Exudative Material and Fragments of Tissue Removed for Biopsy", Am. J. Clin. Path. 32(4): 397-398, 1959 [does not show on-line except that Google Scholar directs to Technical Bulletin of the Registry of Medical Technologists, 29:155-156,1959, (HERE) which does not show the text, Nov. 2016].
  12. Friedland LM, "A Note Frozen Section Tecnic". Am. J. Clin. Path. 21:797, 1951.
  13. Lillie, RD, Histopathologic Technic and Practical Histochemistry, Ed. 2. Philadelphia: Blackston Company 1954, p. 56.
  14. Jones MV & Calabresi PA, "Agar-gelatin for Embedding Tissues Prior to Paraffin Processing", Benchmarks section, BioTechniques 42:569-570, May 2007 (HERE).
  15. Yadav L, Thomas S, Kini U; "Improvised double-embedding technique of minute biopsies: A mega boon to histopathology laboratory." Indian J Pathol Microbiol 58:12-6, 2015, HERE.
  16. Zozumi M, et. al., "New double embedding technique for specimens of endoscopic submucosal dissection using agarose: Comparison with other media", Journal of Clinical Pathology 63(10):904-9, October 2010, HERE.

(posted 24 November 2002; latest addition 11 February 2017)

 
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