ed with the original fixation protocol, 16 (64%) displayed the
incomplete staining artifact (Figure 1A). Of the 35 slides that
were tested with the new protocol, 0 displayed the incomplete
staining artifact (Figure 1B). While using the new stain line protocol,
there were three cases where cartilage detached from the
slide. In all three cases, the corresponding specimen that was
treated with the original stain line protocol did not detach from
the slide. Heating slides with a slide warmer helped sections
adhere to the slide. As a result of this assay, the new fixation
protocol was incorporated at our MMS laboratories. This artifact,
which was regularly found intraoperatively at five separate
MMS laboratories has sustainably resolved.
LIMITATIONS
Cartilage processing was limited by the new fixation protocol. Acetone hardens tissue to the slide, and it is important to note that the section may detach from the slide during the new fixation process. We found that skipping the fixation steps of the stain line (acetone and water) with cartilage-containing tissue was the best method to avoid tissue detachment. Skipping tissue hydration and fixation does not significantly affect morphologic detail.5 Also, we used a plate warmer and positively charged slides, but with less success than skipping the fixation steps altogether. This limitation leaves room for further troubleshooting.
CONCLUSIONS
Tissue fixation is the most important step of the staining process
during frozen section pathology. In our assay, over 60%
of slides treated with the original fixation protocol had incomplete
staining artifact, while 0% of the slides treated with the
new fixation protocol had incomplete staining artifact. Adding
water between Acetone and Hematoxylin as a step of the tissue
fixation process is necessary to produce high quality stained
slides. This alteration in our tissue fixation protocol eliminated
the incomplete staining artifact which improved consistency in
tissue staining, overall slide preparation quality, and histologic
interpretation. Further studies are needed to fully elucidate the
etiology of this staining artifact.
DISCLOSURES
All authors have no conflicts of interest to declare.
ACKNOWLEDGMENTS
We gratefully acknowledge the assistance of Merica Hale,
Dermatopathology Laboratory Manager at the University of
Utah, for creating the stain line graphic and Scott Florell MD, for
the histologic photographs.
REFERENCES
1. Bouzari N, Olbricht S. Histologic pitfalls in the Mohs technique. Dermatol Clin. 2011;29(2):261-72, ix.
2. Desciak EB, Maloney ME. Artifacts in frozen section preparation. Dermatol Surg. 2000;26(5):500-4.
3. Erickson QL, Clark T, Larson K, Minsue Chen T. Flash freezing of Mohs micrographic surgery tissue can minimize freeze artifact and speed slide preparation. Dermatol Surg. 2011;37(4):503-9.
4. Hruza GJ. Mohs micrographic surgery local recurrences. J Dermatol Surg Oncol. 1994;20:573–7.
5. Larson K, Ho HH, Anumolu PL, Chen TM. Hematoxylin and eosin tissue stain in Mohs micrographic surgery: A review. Dermatol Surg. 2011;37(8):1089-99.
2. Desciak EB, Maloney ME. Artifacts in frozen section preparation. Dermatol Surg. 2000;26(5):500-4.
3. Erickson QL, Clark T, Larson K, Minsue Chen T. Flash freezing of Mohs micrographic surgery tissue can minimize freeze artifact and speed slide preparation. Dermatol Surg. 2011;37(4):503-9.
4. Hruza GJ. Mohs micrographic surgery local recurrences. J Dermatol Surg Oncol. 1994;20:573–7.
5. Larson K, Ho HH, Anumolu PL, Chen TM. Hematoxylin and eosin tissue stain in Mohs micrographic surgery: A review. Dermatol Surg. 2011;37(8):1089-99.
AUTHOR CORRESPONDENCE
Adam Tinklepaugh MD adam.tinklepaugh@hsc.utah.edu
