ORIGINAL RESEARCH

Deep Sections, a Guiding Path for Accurate

Diagnosis in Histopathology - A Retrospective
Study
Shankargouda PaƟl*, Roopa Rao†, Amrutha Nagaraja‡
*MDS, Lecturer, †MDS, Professor and Head, ‡Post Graduate Student, Department of Oral and Maxillofacial Pathology,
M.S.Ramaiah Dental College and Hospital, Bangalore, Karnataka, India

A B S T R A C T
Background: The role of pathologists is challenging, as it requires a creative mind to visualize three dimensional aspects of various
tissue specimens which becomes their daily menu. Also, training the histotechnologists becomes part & parcel of their life as their skills
in pampering the tissues decides the fate of the biopsy. Thus, this study emphasizes the significance of deeper sections & training
the technicians. Aim: To determine the number of cases for which deeper sections were required & to emphasize the importance of
deep sections over superficial sections. Materials and Methods: A total of 250 cases were retrieved and reviewed from the files of
our department of oral pathology MSRDC dated back from August 2011 to May 2013. The superficial and deeper sections of individual
selected cases were analysed by two observers. Results: Of 250 cases evaluated, 25 (10%) cases were requested for deeper
sections. Of 25 cases, 11/25 (44%) cases required deeper sections as superficial sections were undiagnostic, 13/25 (52%) cases
also mandated deeper sections as superficial sections mislead the diagnosis and only 1/25 (4%) case exclusively revealed additional
features on deeper sectioning. Conclusion: Deeper sections in some cases provide an array for accurate diagnosis. Though it is a
little time consuming, if not in all cases, a few cases which require deeper sections should be considered without a second thought, as
it can lead to a more appropriate diagnosis.

Keywords: Superficial section, deep section, accurate diagnosis, tissue orientation, plane of sectioning

INTRODUCTION apprehension amongst the concerned doctor, patients &

Any biopsy received by a pathologist would normally
take 48 hours to report. The onus primarily lies on the
pathologist from the time of receiving the biopsied
specimen to its disposal. At times lots of hidden issues
may delay the report further raising the anxiety &
Serial Listing: Print-ISSN (2320-2068)
Online-ISSN (2320-2076)
Formerly Known as Journal of Advanced Dental Research
Bibliographic Listing: Indian National Medical Library, Index Copernicus,
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the attendants.
Pathology report is the summation of information that
describes the diagnosis. It has a key role in decision making
about the treatment & prognosis. The specimen received has
to go through several procedures like grossing, embedding,
sectioning, staining before the details of the tissue are
translated into diagnosis. At times certain situations
demand deeper sections to arrive at final diagnosis. Variety
of sections can be opted based on the need.

SERIAL SECTIONS AND STEP SECTIONS

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Quick Response Code:
Our technicians can prepare slides to display multiple
Website:
www.joaor.org sections from a tissue sample. Collecting every section
creates a serial section; taking sections at specified
DOI: depths from a block (e.g. every 50 microns) creates
***** a step section.[1] Deeper sections are sometimes used
interchangeably with serial and step sections.[2]

Address for correspondence: Shankargouda Patil, Department of Oral and Maxillofacial Pathology, M.S.Ramaiah Dental
College and Hospital, Bangalore, Karnataka, India. E-mail: dr.ravipatil@gmail.com

Journal of Advanced Oral Research / May-Aug 2013 / Vol. 4 No. 2 15

 Patil, et al.
ADVANTAGES OF STEP VERSES SERIAL
SECTIONS
From the standpoint of the quality of information
obtained, serial sections are superior to step sections.
However, by using step sections it is possible to subject
the residual sections for special stains[3] and if needed or
can be retained for future usage.[1]
Bruecks et al. used the time-motion, task analysis
approach to assess the technical labour costs of
prospective step sections verses retrospective step
sections and concluded that the use of prospective step
sections is essentially cost neutral and case turn-around
time is improved by 9-45%.[2]
RETROSPECTIVE AND PROSPECTIVE SECTIONS
Traditionally, deeper levels are obtained, at the request
of the pathologist, after the original slides have
been reviewed-retrospective step sections. In some
laboratories step sections are prepared prior to receipt
of the slides by the histopathologist- prospective step
sections.[1]
Situations that warrant deeper sections are following:
• Initial sections when obtained are superficial and
undiagnostic[2]
• To identify positive areas that could contribute to a
proper diagnosis and rule out certain diagnosis[2,4]
• To avoid diagnostic error and subsequent malpractice
claims[5]
• To enhance sensitivity & diagnostic accuracy[2,6,7]
• Disclosing additional pathological findings &
hidden malignancies[1,2]
• Orientation & plane of sectioning of the tissue
specimen pose problems[8,9]
• If wide disparity is evident on superficial sections
with that of provisional diagnosis
• To know the clearance of surgical margins & the
extent of invasion
• To rule out artifacts
DEEPER SECTIONS ARE NOT THE PREFERENCE
OF CHOICE IN ALL CASES
• Superficial sections are themselves diagnostic most
of the times.
• Wastage of tissue material which may hinder further
investigation
• Loss of archival tissue material
• Technical work load (cutting sections, staining and
labelling of sections.)[1]
16 Journal of Advanced Oral Research / May-Aug 2013 / Vol. 4 No. 2
• Increased supply costs for staining materials, glass
slides, etc., and the costs for an Increased storage
volume must be considered[1]
• Delay in dispatching the report[2]
The studies undertaken emphasizing its importance
is sparse. Currently, standards for handling small
oral biopsy specimens have not been established.
Thus, we undertook this study of retrieving the files of
oral histopathological reports which mandated deep
sections & to know the importance of deep sections over
superficial ones. Also, emphasis is made on training the
technicians & student population with illustrations to
minimize the errors & hasten the work.
MATERIALS AND METHODS
A total of 250 cases were retrieved and reviewed from
the files of our department of oral pathology MSRDC
from the year Aug 2011 to May 2013. The superficial
and deeper sections of individual selected cases were
analysed by two observers.
Inclusion criteria
Cases documented in the register, requested for deeper
sections or for additional features to ascertain the findings.
Exclusion criteria
Those requested for re-staining & special staining.
Also, number of deeper levels required was out of our
purview.
RESULTS
Out of 250 cases evaluated, 25 (10%) cases were
requested for deeper sections. Of 25 cases, 11/25 (44%)
cases required deeper sections as superficial sections
were undiagnostic, 13/25 (52%) cases also mandated
deeper sections as superficial sections mislead the
diagnosis and only 1/25 (4%) case revealed additional
features on deeper sectioning [Graph 1, Figure 1].
DISCUSSION
The uncertainty in patients while waiting for biopsy
report can cause a lot of stress and anxiety. Not knowing
when the results will be ready and not understanding
why testing sometimes takes longer than expected can
cause extra concern. Routine biopsy results may be
ready at the earliest within a day or 2 after the sample
is received in the laboratory.
 Patil, et al.

A B C D E F

a b c d e f
Figure 1: Representing the various lesions where deeper sections were opted. Capital letters indicate superficial sections and small letters indicate deeper sections.
(A) Only epithelium, (a) pemphigoid lesion. (B) Cross section showing ameloblastoma like follicles, (b) verrucous carcinoma. (C) Dentigerous cystic epithelium,
(c) Nasopalatine duct cyst. (D) Haemorrhagic fibrous stroma, (d) Unicystic ameloblastoma. (E) Non keratinized cystic lining with mucous cell metaplasia and connective
tissue wall, (e) Low grade Mucoepidermoid carcinoma. (F) Hyperplastic epithelium, (f) Microinvasive Oral squamous cell carcinoma

as only selective areas are preferred.

c. S p e c i a l s t a i n s , h i s t o c h e m i c a l o r
immunohistochemical stains usually delay results
for another day. Other advanced studies like flow
cytometry, electron microscopy, and molecular
pathology techniques can take even longer,
sometimes days, before results are ready.
d. Getting a second opinion for hard or rare cases from
an expert by overnight mail or as digital images
delays the report for several days.
e. Dispatching of the reports where routine protocol
has to be followed

Training of the technicians

Graph 1: Depicting the percentage of cases which mandated deeper sections
Delay in the biopsy report may be subjected to
various reasons
Pre-laboratory incidents
Almost half the total number of pathology incidents
include problems with taking samples (inadequate
biopsy, damaged specimen during removal,
unrepresentative of the lesion or too small lesions,[2]
improper labelling with no mention of urgency,
preserving samples (Poorly fixed specimen,[2] arrival
in the laboratory (either delayed or not at all).
Laboratory incidents
Technical reasons for delays in reporting are enlisted
below:
a. Hard tissues prolong the processing as they are
subjected to special handling like decalcification
procedure. Fatty tissues, larger samples such as
entire organ or salivary gland require extra time.
b. Need to look at more tissue in case of larger samples
Although grossing forms the first & important step
in tissue processing, specimen orientation & plane
of sectioning are the pivotal & critical steps to decide
the fate of the biopsy. Sectioning tissues is a real art
and takes much skill and practice. Histotechnologists
are the artists of the laboratory. A calm frame of mind
is required, since serial sectioning is labor intensive.
Proper supervision and training is mandatory to avoid
wastage of material & obtain diagnostic sections. This
can be achieved by orienting technicians to inking,
recording the 3D appearance of the specimens & follow
the illustrations depicted in [Figures 2 and 3]. In any
event, deeper sections are the sequential array of many
small sections that expedites the photography.
Reorientation, re-embedding & Plane of sectioning
Correct embedding & orientation is the last step where
grossing can directly have an impact on tissue specimen.
We did encounter few problems with orientation &
plane of sectioning of verrucous growth as superficial
sections revealed deceptive odontogenic islands
misleading the diagnosis, while deeper sections, by

Journal of Advanced Oral Research / May-Aug 2013 / Vol. 4 No. 2 17

 Patil, et al.
I II
Figure 2: (I) Vegetable Broccoli depicting verrucous growth. A to F shows
series of sections which show the impact of sectioning on the interpretation of
the verrucous lesion. (II) Vegetable pear depicting difficulties encountered in
visualizing 3D structures – A to F shows series of sections which show the effect
of sectioning on the interpretation of the lesion
I II
Figure 3: (I) Orange depicting tumour mass. Series of section showing the
appearance of segments of an orange (A to H) that has cut in various planes of
section. A is transversely cut, E is longitudinally cut. Although the segments are
septum bounded compartments with similar dimension, noticeable inequalities
are size are seen in certain planes of section(C, D, G, H). (II) Cooked Pasta
depicting tubular structures (Ex. Ductal structures, blood vessels) Appearance of
sections of a curved tube (A to D) and a straight tube (E to G) at different levels
relative to the centre of the lumen
reorienting the tissue we could arrive at the diagnosis
of verrucous carcinomas in Figure 1 (B, b). According to
Jason W. Nash et al. additional findings were identified
when the tissue blocks are re-embedded and reoriented
and sections taken from the opposite side of the tissue
block.[10]
Problematic lesions
Cystic lesions like dentigerous cyst/OKC or neoplasm
like cystic ameloblastoma possess fragile tissue that
curls during processing. Even with careful orientation,
the sections may not show the complete epithelial
lining. There is no proper guidance for the technician
18 Journal of Advanced Oral Research / May-Aug 2013 / Vol. 4 No. 2
to assess the depth of the tissue, even if diagrammatic
representation or inking of the specimen is available,
and therefore deeper sections are mandatory.[2]
Various studies have suggested opting for deeper
sections in case of incorrect orientation & unclear
histological findings in initial sections.[8,11]
Sampling error
One of our biopsied specimens Figure 1 (D, d) initially
showed haemorrhagic stroma resulting in sampling
error, due to incomplete sectioning through the tissue
block leading to false diagnosis. Later deep sectioning
revealed unicystic ameloblastomatous lining which is
similar to study undertaken by Andrea et al.[4]
Thus, deeper sections are requested in many of the
situations as mentioned above. In the present study such
a requisition was made in those superficial sections that
were undiagnostic representing only a part of biopsy
tissue, accounting to 11 (44%) cases, those which
mislead the diagnosis 13 (52%) cases, & 1 case (4%)
exclusively to rule out additional features although
initial sections were diagnostic. Incidentally, one of the
case enlisted also revealed additional features. Refer
Table 1 for the various reasons quoted.
In oral lesions, the additional sections were beneficial
for appropriate diagnosis for undiagnostic lesions,
confirm and clarify the diagnosis for some difficult cases
and also disclosed some additional features.[2]
In small skin biopsies, step sections provided diagnosis
for nondiagnostic cases, in some cases it even resulted
in a change in diagnosis, while in others it clarified the
diagnosis owing to improved diagnostic accuracy [4,12]&
according to Shubha et al. the step sections improved the
diagnostic accuracy & also discovered hidden malignancies
without compromising on economy & turnaround time.
In a case reported by Betina et al. the ulcerated skin
lesions, the additional sections were made to rule out
certain lesions like bullous disease or of herpes simplex
virus infection but surprisingly showed diagnosis of
superficial basal cell carcinoma thus spared them from
unwanted diagnostic agony.
According to Yoginder et al., in oesophageal or
gastroesophageal junction biopsies, deeper sections
resulted in better evaluation of dysplasia and found
additional findings.
 Patil, et al.

Table 1:The importance of deeper over superficial sections

Group 1: List of superficial sections revealing undiagnostic features (total 11 cases)
Superficial sections revealed Deeper sections revealed Diagnosis No. of cases
Only epithelium Epithelium and connective tissue along with Pemphigoid lesion 1
the sub epithelial split
Only fibrous tissue Psamomma like ossification Psammomatoid ossifying fibroma 1
Only fibrous tissue 2-3 layers of reduced enamel like cystic lining Dentigerous cyst 2
Tissue tear with loss of epithelium Epithelium obtained with dysplastic features Epithelial dysplasia 1
Only fibrous tissue Cystic lining with ameloblast and stellate Unicystic ameloblastoma 3
reticulum like cells
Only salivary gland tissue Cystic lining with mucin and mucinophages Mucocele 1
Only inflamed fibrous tissue Cystic lining with ameloblast, stellate Inflamed unicystic 1
reticulum like cells and inflamed stroma ameloblastoma
Only fibrous tissue and a bit of Ciliated cystic lining Nasopalatine duct cyst 1
noncharacteristic epithelium
Group 2: List of superficial sections that mislead the diagnosis (total 13 cases)
Superficial section resembled Deeper section Diagnosis No. of cases
Trabecular ossifying fibroma Psammoma like calcifications Psammomatoid ossifying fibroma 1
Solid ameloblastoma Cystic lining with ameloblast and stellate Unicystic ameloblastoma 2
reticulum like cells
Lateral periodontal cyst Cystic lining with ameloblast and stellate Unicystic ameloblastoma 1
reticulum like cells
Dentigerous cyst Ciliated cystic lining Nasopalatine duct cyst 1
Ameloblastoma-cross section Finger like papillary projections, parakeratin Verrucous carcinoma 1
plugging, elephant foot like rete ridges
Oral submucous fibrosis Dysplastic epithelial islands invadingstroma Oral squamous cell carcinoma 1
Hyperplastic epithelium Invading epithelial cells in the fibrous stroma Microinvasive oral squamous cell 1
carcinoma
Pemphigus No intraepithelial split noted 1
Verrucous carcinoma Invading epithelial cells in the stroma Microinvasive oral squamous cell 1
carcinoma
Oral squamous cell carcinoma Artifactual epithelial island as island merged Epithelial dysplasia 1
with the overlying epithelium
Unicystic ameloblastoma Mural proliferation with granular cell change Can be considered as solid 1
ameloblastoma
*Nonkeratinized cystic lining with mucous Microcyst and macrocyst with island of Low grade mucoepidermoid 1
cell metaplasia and connective tissue wall epidermoid and mucous cells carcinoma
Group 3: List of additional features encountered on deeper sections (total 1 cases)
Superficial section Deeper section with additional findings No. of cases
Keratocystic odontogenic tumor Cholestrol clefts and Asteroid bodies 1
Nonkeratinized cystic lining with mucous Cholestrol clefts
cell metaplasia and connective tissue wall*
*This case, apart from misleading the diagnosis, also revealed additional features in deeper sections

In gastrointestinal pathology, the diagnostic efficacy
of additional step sections in colorectal biopsies
originally diagnosed as normal, yielded new diagnostic
information such as tubular adenoma & lymphocystic
colitis in deeper sections.[13]
In Transbronchial Lung Biopsy specimens, Step
sections were especially useful for the detection of
epitheloid granulomas and tumor tissue in patients
with sarcoidosis and lymphangiosis carcinomatosa,
respectively, hence, overall the step sections were
considered to have clinical impact.3
In a study evaluated by Jason et al. relating to colorectal
biopsy specimens, the deeper levels showed neoplastic
findings in some benign cases which drastically changed
the diagnosis and treatment.
In a retrospective study by Yehung on cerival biopsy
specimens, step sections changed the diagnosis
and hence was a considered useful in providing an
appropriate diagnosis.
Brueks et al. analyzed the utility of prospective step
section and turnaround time and concluded that the use
of prospective step sections is essentially cost neutral
and case turn around time is improved by 9-45%.
CONCLUSION
This study highlights the essential utility of deeper
sections in improving the diagnostic accuracy in
routine oral pathology practice without compromising
the economy & case turnaround time. Also, hidden
malignancies affirm the potential benefits of deep

Journal of Advanced Oral Research / May-Aug 2013 / Vol. 4 No. 2 19

 Patil, et al.
section analysis. We recommend the utility of deeper
sections in specific oral lesions to be evaluated further.
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Cite this article as: Citation will be included before issue gets online***
Source of Support: Nil. Conflict of Interest: None delcared.