Quantitative and qualitative comparison of two routine staining protocols for Mohs micrographic surgery Jeave Reserva MD 1 ,Cindy Krol BS 1 , Lauren Moy MD 1 , Reeba Omman MD 2 , Dariusz Borys MD 2 , Jodi Speiser MD 2 , Kristin Lee MD 1* 1 Division of Dermatology and 2 Department of Pathology, Loyola University Medical Center, Maywood, IL ABSTRACT RESULTS CONCLUSION REFERENCES INTRODUCTION METHODS 1. Larson K, Ho HH, Anumolu PL, Chen TM. Hematoxylin and eosin tissue stain in Mohs micrographic surgery: a review. Dermatol Surg. 2011 Aug;37(8):1089-99. 2. Gray A, Wright A, Jackson P, Hale M, Treanor D. Quantification of histochemical stains using whole slide imaging: development of a method and demonstration of its usefulness in laboratory quality control. J Clin Pathol. 2015 Mar;68(3):192-9. 3. Humphreys TR, Nemeth A, McCrevey S, Baer SC, Goldberg LH. A pilot study comparing toluidine blue and hematoxylin and eosin staining of basal cell and squamous cell carcinoma during Mohs surgery. Dermatol Surg. 1996 Aug;22(8):693-7. Background: Although prior studies have investigated novel alternatives and adjuncts to hematoxylin and eosin (H&E) stains for Mohs Micrographic Surgery (MMS), to the best of our knowledge, no formal studies have compared H&E staining protocols using both quantitative and qualitative methods. Objectives: Investigate the effect of a shortened staining protocol on the quality of MMS sections Methods: Fourteen MMS sections of similar tissue thickness and size were selected and stained using a standard and an abbreviated staining protocol. Whole slide images were obtained and subsequently analyzed using ImageJ color deconvolution. In addition, a dermatologist graded each case’s overall readability on a 1 to 4 scale (with 4 being best quality). Results: Statistically significant differences (p<0.05) between the two staining protocols’ mean H&E intensities and H:E ratios were observed. The abbreviated and standard protocol’s median overall readability were 3 and 4, respectively, with significant differences in their distributions (Mann–Whitney U=20.5, p<.05 two tailed). Conclusions: The shortened staining protocol has an acceptable, albeit less than ideal, readability and may be of value in specific clinical scenarios and practice settings. Frozen tissue slides of similar tissue size from MMS cases were selected by the Mohs histotechnologist for staining using conventional staining method or the abbreviated H&E staining method. Both protocols use a Shandon Linistat Linear Stainer with time per cup of 20 seconds and 5 seconds between cups. The standard H&E protocol uses acetone (cup 1), tap water (cup 2), Gill’s Hematoxylin III (cup 3), tap water (cup 4 -7), absolute alcohol (cup 8), eosin Y 1% and absolute alcohol in 1:1 ratio (cup 9), absolute alcohol (cups 10-13), and histoclear (cups 14-17). The abbreviated H&E staining protocol is the same as above with the elimination of 2 waters, 2 alcohols, and 1 clearant. The selected slides were electronically scanned using whole slide imaging (Aperio CS2, Leica Biosystems Inc., Ontario, Canada) and color quantified by the ImageJ program, a free image analysis software developed by the National Institutes of Health (NIH). 2 ImageJ color deconvolution was used to separate H&E staining of images and analyze its color intensity. A blinded dermatologist graded each case’s overall readability on a 1-4 scale (with 4 being best quality). 3 Mean hematoxylin and mean eosin intensities were compared to determine significant differences between the staining protocols. Mann-Whitney test was used to compare median overall readability between the two staining protocols. A total of fourteen MMS cases were included in the study. The abbreviated protocol had a mean eosin intensity of 7.69 x10 -3 (+ 7.98 x10 -4 ) and hematoxylin intensity of 1.81 x10 -2 (+ 1.4 x10 -2 ). This was statistically significantly different (p<0.05) from our standard protocol’s mean H&E intensity [9.36 x10 -3 (+ 2.14 x10 -3 ) and 5.29 x10 -3 (+ 7.98 x10 -3 )]. *= CDS Sponsor Disclosure Statement: All authors have no relevant financial conflicts of interest to disclose. Despite significant differences in H&E intensities causing eye fatigue and hindering visualization of lymphocytes and tumor islands, the shortened protocol has an acceptable, albeit less than ideal, readability and may be of value in specific clinical scenarios and practice settings. RESULTS Mean of standard and abbreviated protocol’s H:E ratios were 2.19+ 1.10 and 1.76+ 0.29, respectively. The abbreviated and standard protocol’s median overall readability were 3 and 4, respectively, with significant differences in their distributions (Mann–Whitney U=20.5, p<.05 two-tailed). Figure 1. Representative sections of standard ( top) and abbreviated (bottom) staining protocols. ImageJ color deconvolution (right column) highlights the higher intensity of hematoxylin and eosin staining in the abbreviated protocol. When present in the deep margin, tumor islands (left column) are more apparent in the standard staining protocol. STANDARD SHORT H&E Ratio Figure 2. There was more variability in the H&E staining with the abbreviated staining protocol in comparison with the standard protocol. The success of MMS depends largely on high quality frozen tissue sections. Herein, we investigate how conventional and abbreviated H&E staining protocol affects cellular and stromal tissue components in keratinocyte carcinomas and quantify H&E ratios using computer-assisted image analysis of whole slide images