The presence of metastatic carcinoma in axillary lymph nodes is the most important prognostic indicator in breast cancer and provides crucial staging information¹. Adjuvant therapy is delivered more intensely in node positive than in node negative cases. Low grade small tumors may not get adjuvant therapy when node staging is diagnosed as negative. False negative node reports are not cost effective for the patient. The ADASP has published professional recommendations for complete processing and reporting lymph node specimens submitted for evaluation for metastatic disease². The standard (with exceptions) is that completion axillary node dissection is indicated when sentinel node metastases are found³. Therefore it is essential to demonstrate a metastasis whenever present. A number of retrospective studies performing amplified re-processing of archival cases originally designated negative "by routine processing"  have shown a conversion from node-negative to node-positive cases on the order of 20-25%. ^{1, 4, 5, 6}

We have developed a simple technique for providing slide-based documentary assurance to histotechnologists at time of stepsectioning and to the pathologist during case sign-out that lymph nodes processed according to ADASP standards have been truly sampled at multiple levels as intended.

Following overnight fixation (we use Hartmann's fixative...alcoholic formalin & acetic acid), specimens are thoroughly dissected in order to recover all lymph nodes 1-2 mm in diameter or larger. Unless visibly positive, larger nodes are cross-sectioned at 2-3 mm intervals and entirely processed and small nodes bissected or whole. A trilayered agar depth stick of different-colored layers (orange, yellow, and blue) is placed within an aggregate of small nodes or node cross sections, which are then agar pre-embedded and placed into routine processing cassettes. Further details of this process⁷ are posted long-term on our website⁸. These blocks are then placed in with the routine tissues for overnight tissue processing in our general pathology lab. The specimens are embedded in routine fashion. When the histotechnologist mounts the block into the microtome, the easily visible orange color of the "down" end (at the block face) of the agar depth stick clues the histotechnologist to section according to our "intense lymph node processing protocol". Slides are cut for H&E staining from each third of the entire thickness of the blocked node aggregate. A slide is taken from the middle or central third of the block for immunostaining for pan-keratin antigen as a visibility-enhancing completion survey for occult malignant epithelial ITCs.

It is a prevailing belief that a step-cutting process for the multiple lymph nodes of an axillary node dissection is a prohibitively expensive process. Cost studies may paint a discouraging picture because they fail to consider that any increase in cost is an incremental increase rather than a new increase; therefore, the incremental cost is much less¹⁰. Because the embedded agar depth stick changes color each third of the way through the block, our histotechs have found that this "intense lymph node processing protocol" requires no more than 2-3 minutes per block. That is, the presence of the agar depth stick marker allows the histotechnologist to turn the microtome wheel very rapidly, proceeding from first third, to the middle third, and to the deep third of the paraffin block quickly and without hesitation.

Utilizing this process from 14 October 2002 through 13 October 2003, we seven pathologists interpreted 135 breast cases with nodes and found 45 of those cases (33%) to have one or more lymph nodes containing metastatic tumor. Upon review of each of those cases, it is the author's opinion (similar to the conversion rate of the study which inspired our technique⁹) that as many as 12 (27%) of those 45 cases might have been missed by our previous routine sections and about 4 of those 12 (9% of all positive cases) missed by a microtome sectioning process which depended upon routine estimates of levels by the histotechnologists rather than a controlled and documented process that quickly and methodically forces one to sample at three or more spaced paraffin-block depth levels of the lymph nodes.

Our group has used agar pre-embedding since 1978, and we are convinced that the discipline that a multicolored marker embedded in the block gives to the microtoming process easily allows a cost effective, more thorough, life-saving exam of nodes at only a mildly increased investment in technologist and pathologist time. We offer the process in hopes that one or more major centers will test it and confirm or refute its value. Most importantly, oncologists (as ours do) can rest more assured that a "node negative" diagnosis by this method has a significantly increased likelihood that the diagnosis is a "true-negative" diagnosis. In the meantime, we have added a fourth H&E slide at the point of disapparance of the colored agar stick, deep within the block. And, we are attrac ted to the idea of low powered microscopic exam of the fressh cut surfaces of the SLN¹³ prior to FS and with a rapid touch prep exam as a means of in initial morphological assessment with waiver of FS if negative.

Bibliography:

1. Yared MA, et. al., "Recommendations for sentinel lymph node processing in breast cancer", Am J Surg Pathol 2002; 26(3):377-382.
2. Association of Directors of Anatomic and Surgical Pathology. ADASP recommendations for processing and reporting lymph node specimens submitted for evaluation of metastatic disease. Am J Surg Pathol 2001; 25:961-3.
3. Van Zee KJ, et.al., "A Nomogram for Predicting the likelihood of Additional Nodal Metastasis in Breast Cancer Patients with a Positive Sentinel Node Biopsy", Ann Surg Oncol 2003; 10(10):1140-1151.
4. Fisher ER, et.al., "Detection and significance of occult axillary node metastases in patients with invasive breast cancer." Cancer 1978; 42(4):2025-31.
5. Hanna WM, et.al., "Pathologic Characteristics of Breast Cancer that Predict for Local Recurrence After Lumpectomy Alone." Breast J 1999; 5(2):105-111.
6. Cote RJ, et.al., "Role of immunohistochemical detection of lymph-node metastases in management of breast cancer. International Breast Cancer Study Group." Lancet 1999; 354(9182): 896-900.
7. Shaw EB, et. al, "Correct orientation of specimens for histologic processing: Preliminary embedding in agar." Am J Dermatopathol 1983; 5(2):165-167.
8. Pathology Associates of Lexington, P.A. website http://www.palpath.com/agards.htm, accessed 25 March 2004.
9. Tan LK, Giri D, Panageas K, Cranor M, Bevilacqua J, Hummer A, Brogi E, Norton L, Hudis C, Borgen P, Memorial Sloan-Kettering Cancer Center, New York, NY, "Occult/micrometastases in axillary lymph nodes of breast cancer patients are significant: a retrospective study with long-term follow-up", presentation at the 2002 ASCO Annual Meeting, http://www.asco.org/ac/1,1003,_12-002636-00_18-0016-00_19-00146,00.asp, accessed 26 April 2004.
10. Weaver, DL, et.al., "Sentinel Lymph Nodes and Breast Carcinoma" AJSP 2003; 27(6): 842-845.
11. Taylor CR & Cote RJ, Immunomicroscopy: A Diagnostic Tool for the Surgical Pathologist, 2nd Ed. 1994 (#19 in the series of Major Problems in Pathology), pages 233-235.
12. Diaz NM, et. al., "Benign Mechanical Transport of Breast Epithelial Cells to Sentinel Lymph Nodes", AJSP 28(12) :1641-1645, December 2004.
13. Varga Z, et. al., "Use of intraoperative stereomicroscopy for preventing loss of metastases during frozen sectioning of sentinel lymph nodes in breast cancer", Histopathology 52(5):597-604, April 2008.