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3 The Role of Laboratory Diagnosis in Oral and Maxillofacial Pathology: An Overview
32
Figure3.3 Orientated en- bloc left neck dissection.
As part of patient treatment, a selective neck
dissection is often also completed alongside the
main resection specimen due to the risk of
metastasis to loco- regional lymph nodes. Neck
dissections can be received en bloc (as per
Figure2.3) with the individual neck levels
identified by the surgeon using numbered tags
(identifying the neck level). The surgeon may
submit the levels in separate specimen pots.
one can also take photographs of the specimen after it has been inked so the pathologist can
identify how each margin has been colour- coded. These photographs can be labelled at cut- up
(e.g.anterior view) with relevant excision margins annotated with an arrow (e.g. buccal margin
[red ink]).
Once a pathology report has been written for the specimen, the pathologist can annotate the
photographs to highlight any involved margins or prognostic information. This can be shared with
the surgeon and the rest of the clinical care team to help describe the salient features of the case.
Radiographs can be used to assess specimens involving bone. This can be useful when no
pre- operative imaging of the case is available, and one wants to determine the extent of bone
invasion before cutting up the specimen. It can also be used to monitor the decalcification
process once the bone has been put in acid– the opacity of the bone becoming progressively less
radio- opaque over time(6).
3.7 Microscopic Assessment
3.7.1 Haematoxylin– Eosin Staining andSpecial Stains
The microscopic assessment of a biopsy usually begins with examining the haematoxylin– eosin
(H&E)- stained slide. From viewing this initial slide, the decision can be taken whether there is
sufficient evidence to provide a diagnosis or whether additional stains or testing are required.
The slide should first be examined at low power to ascertain the amount of tissue present and its
basic structure– is it fragmented, has mucosa and what are the basic tissue types present? Adopting
a systematic approach is essential so each part of the slide is viewed and nothing is missed. Next,
the pathologist will usually examine the slide in more detail using the higher- magnification lenses,
focusing on areas of interest. At this point, the pathologist may be able to see what type of pathology is present or the basic disease process, e.g. is the disease process inflammatory or neoplastic?
If there is insufficient tissue present on the slide, but there is thought to be more tissue in the block,
the pathologist can ask for a ‘deeper’ section to examine more tissue. Similarly, a pathologist may
ask for ‘levels’ to examine a region of interest in more detail.
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3.7 Microscopic Assessment
(a) (b)
Figure3.4 Examples of special stains used in oral pathology. (a) PASD staining highlights the presence of
fungal hyphae in the superficial keratin layers of this oral biopsy specimen. (b) An EVG stain can highlight
the elastic fibres in the elastic lamina of blood vessels. The arrow indicates an example.
Table3.1 Some ofthe commonly used special stains andtheir uses.
Stain Use
33
Periodic acid Schiff
(PAS)
Periodic acid Schiff
with diastase (PASD)
Congo Red To detect amyloid, which will stain red and show apple- green birefringence under
Grocott To detect fungal organisms which stain black
MSB (Martius
Scarlett Blue)
Perls To detect iron deposits which stain blue. It can confirm the presence of
Van Gieson To detect elastic fibres which stain black. It can help assess vasculitis as the elastic
To detect mucins, glycogen, and basement membrane. For example, zymogen
granules in an acinic cell carcinoma would be positive and stain magenta
The diastase removes glycogen. This can be useful in differentiating glycogen
from mucin.
polarised light.
A trichrome stain is used to assess tissue deposits, particularly fibrin. Fibrin stains
red, collagen blue and erythrocytes yellow
haemosiderin (containing iron) versus melanin (negative reaction)
lamina will be incomplete owing to inflammatory damage to the vessel wall
Several ‘special stains’ are available to the pathologist to help assess the pathology further and
refine a diagnosis(7) (Figure3.4). Some of the more commonly used stains and their uses are given
in Table3.1.
3.7.2 Immunofluorescence
Immunofluorescence is a technique using light microscopy and a fluorescent microscope. In histopathology, direct immunofluorescence on fresh tissue detects the specificity of fluorescently
tagged antibodies to their target antigens in biopsies from mucosa and skin. Diagnosis of several
auto- immune blistering conditions requires direct immunofluorescence in addition to light
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3 The Role of Laboratory Diagnosis in Oral and Maxillofacial Pathology: An Overview
(a) (b)
34
Figure3.5 (a) An example of the intercellular IgG pattern (chicken wire pattern) is seen in Pemphigus
vulgaris. (b) This example demonstrates the linear IgG pattern at the basement membrane in mucous
membrane pemphigoid.
microscopy. For example, in pemphigus vulgaris, the H&E slide would show acantholysis of the
epithelium, while the direct immunofluorescence would show fluorescence for intercellular IgG
and C3 (chicken wire pattern) (see Figure3.5).
3.7.3 Immunohistochemistry
IHC is another tool in the pathologists’ armamentarium. It can be a valuable adjunct to aid
diagnosis, especially in poorly differentiated tumours or metastatic disease from an unknown
primary site. IHC uses antibodies to indicate if particular antigens are present within the tissue
sample. Awide range of IHC tests are available, and broad categories of tumour differentiation
and appropriate tests are described in Table 3.2. The pattern of staining encountered can vary
according to the location of the antigen within the cell (i.e. stains may be expressed in the nucleus,
cytoplasm or on the cell membrane) (Figure3.6). Therefore, it is essential to know the site where
the stain should be expressed(8). If staining is positive in the wrong side of the cell, it may be
misinterpreted as a positive result and thus may lead to an incorrect diagnosis.
Whilst some IHC markers are specific, others may be expressed in several very different neoplasms
(e.g. diffuse S100 expression can be seen in a benign schwannoma but also in malignant melanoma).
For this reason, pathologists often adopt a panel- based approach to IHC to ensure the patterns of IHC
marker expression are interpreted in the correct context and always compare these results with H&E
features to arrive at the correct diagnosis. As well as used in the diagnosis of tumours, IHC can also be
used to give an overview of the prognosis of a tumour. Ki67 (or MIB1) is a general proliferation marker
with higher expression in malignant disease processes. Other markers, such as ER and HER2, can be
used as a predictive marker for breast carcinoma. IHC can also screen a patient’s suitability for certain
immunotherapy treatments. Immunotherapy is more frequently used in the management of head and
neck cancer patients, especially in those with advanced disease at presentation and in those with
recurrence following surgical and other therapeutic treatments. IHC for PD- L1 can be performed on
tumour samples in the head and neck. Expression is deemed positive if staining is present in the cell
membrane of tumour cells or surrounding inflammatory cells, and a combined positive score is calculated to determine if a patient would be suitable for treatment(9).
IHC has multiple indications, and a brief overview has been discussed.
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Table3.2 Examples ofIHC used fortumour differentiation determination.
Type IHC test
Epithelial Cytokeratins, e.g. AE1/AE3, MNF116, Cam5.2, CK5/6, CK7, CK20, EMA
Myoepithelial S100, SMA, p63, calponin
Melanocytic S100, Melan- A/MART- 1, HMB45, SOX10
Mesenchymal Vimentin
Endothelial CD31, CD34, Factor VIII
Neural S100, SOX10
Smooth Muscle SMA, desmin, calponin, SMM, h- caldesmon
Skeletal Muscle Desmin, myogenin, Myo- D1
Neuroendocrine Synaptophsin, Chromogranin, CD56, INSM1
Proliferation marker Ki67 (MIB1)
Haematopoietic CD45: pan- haemopoietic marker
CD20: pan- B cell marker
CD3: pan- T- cell marker
CD138: pan- plasma cell marker
Markers associated with
specific organ tumours
Markers associated with
viruses and bacteria
Prognostic markers p16 (surrogate maker of HPV infection), PDL1
TTF1 (lung/thyroid), thyroglobulin (thyroid), ER (breast/ovarian/
endometrial and others), PR (breast/ ovarian/endometrial and others), PSA
(prostate), CDX2 (colorectal/bladder)
HPV, EBER (EBV), Treponema Pallidum (syphilis)
35
3.8 Molecular Testing andCytogenetics
As molecular techniques have developed over the last century and our understanding of the
molecular landscape of tumour biology has broadened, we now have a greater awareness of
tumours with unique genetic alterations. Several different techniques are available to investigate
the presence of genetic alterations in tumour samples, and the most commonly adopted methods
include fluorescent in situ hybridisation (FISH), in situ hybridisation (ISH), polymerase chain reac-
tion (PCR) and next- generation sequencing (NGS). Some of the mutations and rearrangements
that these methods can identify are ‘tumour defining/specific’ and thus diagnostic of specific
tumour entities (i.e. MEF2C- SS18 fusion in microsecretory adenocarcinoma of the salivary glands).
This has allowed for the discovery of new entities. Therefore, pathologists can subtype tumours
instead of assigning tumours to a carcinoma ‘not otherwise specified’ (NOS) category. This will
allow for better prognostic information to be collated about these tumours so that future generations will better understand the tumour behaviour and be able to adopt the most appropriate treatment modalities.
Although some genetic alterations are tumour- specific, others can be found in several unrelated
tumours (i.e. BRAF v600E can be found in skin melanoma but may also be identified in ameloblas-
toma). Similar to IHC, the findings of molecular and cytogenetic tests must be considered alongside the H&E morphology and IHC findings before a final diagnosis is achieved. An integrated
report should be issued linking all investigations. The discovery of these genetic alterations also
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3 The Role of Laboratory Diagnosis in Oral and Maxillofacial Pathology: An Overview
(a)
(c)
(b)
36
Figure3.6 Examples of how IHC is used in diagnostic practice. (a) An H&E stain shows a core biopsy of a
neoplasm within the parotid gland. (b) p63 IHC shows diffuse nuclear expression in the tumour, whereas
(c)S100 shows diffuse nuclear and cytoplasmic expression. In this context, the expression of both P63
andS100 demonstrates a myoepithelial cell- rich neoplasm, including a pleomorphic adenoma and
myoepithelioma in the differential diagnosis.
opens the possibility for targeted gene therapies as a treatment modality for several different
tumour types; for example, targeted therapies are available to patients with melanoma if a BRAF
v600E mutation is identified within their tumour sample(10).
3.9 Conveying Diagnoses andUncertainty: TheLimitations
ofaDiagnosis
When writing pathology reports, the objective is to describe the salient microscopic features and
provide a precise diagnosis. Having the diagnosis on a separate line in the report in bold type or
capital letters makes it easy for the clinician to find. Suppose the clinician has offered a provisional
diagnosis on the pathology request form. In that case, it is helpful for the pathologist to note in the
conclusion of their report whether the entity was present or not, e.g. There is no evidence of granu-
lomatous inflammation. Similarly, stating any ‘negative findings’ means the clinician can see precisely what has been excluded, e.g. There is no evidence of candida, dysplasia or malignancy in the
material examined.
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References
It is not uncommon for the pathologist to be unable to give a specific diagnosis. This can result
from insufficient tissue from examination, non- specific features in the histology or because there
is considerable overlap between disease processes. However, it is essential to avoid unnecessary
ambiguity, for example, by including excessive caveats to the diagnosis or unclear language, as the
report must be helpful to the clinician.
There are often degrees of certainty ascribed to the diagnosis by the pathologist, and being able
to communicate this to the clinician is essential. For example, pathologists often use phrases
suchas ‘the features are those of ’, ‘the features are consistent with’, or ‘the features are suggestive of ’,
indicating increasing levels of uncertainty(11). A simple email or phone call to the clinician
cansupplement the pathology report and express any hesitation with the diagnosis. Reviewing
previous pathology reports and slides can be a helpful exercise in several circumstances, such as
(i)when the latest biopsy is discordant with the previous biopsy of the same lesion, (ii) when a
disease process is evolving, and one wants to compare its appearance over time and (iii) when different pathologists have been involved in the diagnoses, and there is a difference in interpretation.
Reviewing the previous pathology can inform the interpretation and wording of subsequent
reports and hopefully avoid confusion.
Pathologists must explain why a specific diagnosis has not been possible and be able to communicate and document the findings in an easy- to- understand format to service users. Guidance and
recommendations regarding additional tests can also be provided, or referrals can be made to other
specialist teams if their input may benefit patient care and management. In addition, results from
different investigations must be reviewed alongside the pathology report if the diagnosis is unclear
(see Section 1.3), and multi- disciplinary meetings can help discuss complicated diagnoses and
patient management(1).
37
3.10 Summary
This chapter has provided an overview of the principles and importance of histopathological
assessment in the oral and maxillofacial setting. The basic macroscopic principles and the
available adjuncts to aid microscopic assessment have been discussed. The pathologist’s role has
also been highlighted alongside the importance of clinicopathological correlation. One must
remember that histopathological assessment is only part of the spectrum of investigative procedures available when assessing oral and maxillofacial complex conditions. Thus, interpretation
alongside other relevant tests and discussion with clinicians is often helpful to ensure patients
receive the correct treatment. Additional relevant reading is listed in the references for the
chapter.
References
1 De Felice F, Tombolini V, de Vincentiis M, Magliulo G, Greco A, Valentini V, etal. Multidisciplinary
team in head and neck cancer: a management model. Medical Oncology. 2018;36(1):2– 6.
2 Thompson LDR. Small round blue cell tumours of the sinonasal tract: a differential diagnosis
approach. Modern Pathology. 2017;30(1):S1– S26.
3 The Royal College of Pathologists. Cancer Datasets and Tissue Pathways. [Internet]. [Cited 2023,
August 23]. Available from: https://www.rcpath.org/profession/guidelines/cancer- datasetsand- tissue- pathways.html
t.me/Dr_Mouayyad_AlbtousH
3 The Role of Laboratory Diagnosis in Oral and Maxillofacial Pathology: An Overview
38
4 International Collaboration of Cancer Reporting. Published Datasets. [Internet]. [Cited 2023,
August 23]. Available from: https://www.iccr- cancer.org/datasets/published- datasets/
5 Brierley JD, Gospodarowicz MK, Wittekind C. TNM Classification of Malignant Tumours,
th
edition. Chichester, England: Wiley Blackwell; 2017.
8
6 Cook DJ, Warren PJ. Cellular Pathology: An Introduction to Techniques and Applications,
rd
edition. Banbury, England: Scion Publishing Limited; 2015.
3
7 Survarna SK, Latyon C, Bancroft JD. Bancroft’s Theory and Practice of Histological Techniques,
th
edition. Amsterdam: Elsevier; 2019.
8
8 Rekhtman N, Baine MK, Bishop JA. Quick Reference Handbook for Surgical Pathologists,
nd
edition. Switzerland: Springer Nature; 2019.
2
9 La Placa CJ, Vilardo MD, Watts BM, Polewski MD, Tabuena- Frolli S, Jansson M, etal. Development
of a companion diagnostic PD- L1 immunohistochemistry assay for pembrolizumab therapy in
head and neck squamous cell carcinoma. Journal of Cancer Treatment and Diagnosis.
2021;5(1):9– 17.
10 Proietti I, Skroza N, Michelini S, Mambrin A, Balduzzi V, Bernardini N, etal. BRAF inhibitors:
molecular targeting and immunomodulatory actions. Cancers. 2020;12(7):1– 13.
11 Stephens- LaBorde I, Brierley DJ. Why won’t the pathologist give me a diagnosis? Interpreting
uncertainty in head and neck pathology reports. British Journal of Oral and Maxillofacial Surgery.
2022;60(5):577– 83.
t.me/Dr_Mouayyad_AlbtousH
Section 2
Homeostasis andCellular Pathology
39
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4
Homeostasis andInfluences ofEnvironment andNutrition
A. V. Chalapathi Rao1 and S. R. Prabhu
1
Department of Pathology, The University of the West Indies, St. Augustine, Trinidad and Tobago
2
School of Dentistry, University of Queensland, Brisbane, Queensland, Australia
2
4.1 Introduction
Cells are the basic living units of the body, which together form tissues and organs that constitute
a functional organisation of the human body. Each cell type is endowed with specific functional
and structural properties and has variable levels of capacity to renew. About 60% of the human
body is composed of fluid, mainly watery solution containing ions, gases, nutrients, chemicals and
waste products. The constituents of intra and extracellular fluids differ significantly and are maintained by unique mechanisms(1, 2).
41
Homeostasis (In Greek: Homeo means ‘Similar’ and Stasis- ‘Standing still’, which refers to staying
the same or unperturbed), the word coined by American Physiologist Walter Bradford Canon in
1926 is essential to the dynamic process by which a living organism maintains a stable internal
environment despite changes in the external conditions. It is vital for the proper functioning and
survival of cells, tissues and entire organisms. Homeostasis allows physiological mechanisms to
adapt, respond to a variety of challenges and carry out their functions optimally(1, 3– 6).
Internal Environment: Extracellular fluid is an essential nectar the cells need to maintain and sus-
tain optimally functional life. Hence, it is termed an ‘Internal Environment’ or ‘Milieu Interieur’
by French physiologist Claude Bernard in the 19th century. Extracellular fluid is everywhere in
the body, which includes plasma and interstitial fluid between the cells and is in continuous
motion. Cells can perform their regular functions as long as the proper concentrations of various
components of extracellular fluids are maintained. The maintenance of constancy in the Internal
Environment is essentially contributed by multiple body organs and tissues. For example, the
lungs are the source of oxygen and also eliminate carbon dioxide, the kidneys play an essential
role in regulating ion concentration, and the gastrointestinal tract provides nutrients and facilitates the elimination of waste products(1, 2, 6, 7).
Key aspects of Homeostasis and Examples:
1) Dynamic Internal Equilibrium wherein there is continuously regulating, monitoring and main-
taining parameters like temperature, pH, blood pressure, ion concentrations, nutrients and
waste products within normal ranges conducive to cellular/tissue function(1, 2, 4).
Pathological Basis of Oral and Maxillofacial Diseases, First Edition. Edited by S. R. Prabhu, Syed Ali Khurram,
Omar Kujan and Merva Soluk Tekkesin.
© 2025 John Wiley & Sons Ltd. Published 2025 by John Wiley & Sons Ltd.
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