Quantitative and qualitative comparison of two routine staining protocols

for Mohs micrographic surgeryJeave Reserva MD1,Cindy Krol BS1, Lauren Moy MD1, Reeba Omman MD2, Dariusz Borys MD2, Jodi Speiser MD2, Kristin Lee MD1*

1Division of Dermatology and 2Department 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 SponsorDisclosure 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.

STANDARDSHORT

H&

E R

atio

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