Quantitative and qualitative comparison of two routine ...
Transcript of Quantitative and qualitative comparison of two routine ...
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