HISTOPATHOLOGIC AND CYTOLOGIC TECHNIQUES
LESSON
BASIC PATHOLOGY
C. REPASO
I. CYTOLOGY
● Cytology (cytopathology) involves examining cells from
bodily tissues or fluids to determine a diagnosis.
● Uses for cytology tests include:
ADVANTAGES DISADVANTAGES
Simple Interpretation is based on cells
Rapid Not a final diagnosis
Inexpensive Cannot determine size and
No or minimum injury during type of lesions
collection
A. Interventional cytopathology
● a pathologist carries out minimally invasive procedures on
palpable masses or masses identified by ultrasound, using a
fine caliber needle (fine needle) to obtain the sample.
● Fine-needle aspiration (FNAB) - most common type of
intervention cytology.
● Other samples
➔ Fluid-filled lumps (cysts) under your skin.
➔ Solid lumps (nodules or masses) under your skin.
➔ Your lymph nodes.
➔ Your pericardial fluid, which is the fluid in the sac around
your heart.
➔ Your pleural fluid
● Other uses for cytology tests include:
➔ To diagnose infectious diseases.
➔ To diagnose inflammatory conditions.
➔ To examine thyroid lesions.
➔ To diagnose diseases involving certain body cavities
B. Exfoliative cytology
● a branch of cytology in which the cells that a pathologist
examines are either “shed” by your body naturally or are
manually scraped or brushed (exfoliated) from the surface of
your tissue
➔ Gynecological samples: A Pap smear, which involves
brushing off cells from your cervix using a swab, is the
most well-known type of exfoliative cytology.
➔ Gastrointestinal tract samples: brush off cells from the
lining of your gastrointestinal tract (your stomach and
intestines) during an endoscopy procedure for cytology
testing.
➔ Skin or mucus samples: scrape off cells from your skin or
mucous membranes, such as the inside of your nose or
mouth, for cytology testing.
● Examples of exfoliative cytology that involve collecting
tissues or fluids that your body naturally sheds include:
➔ Respiratory samples: fluids such as spit and mucus (also
called phlegm or sputum) that you cough up for a
respiratory cytology test.
➔ Urinary samples: collect a urine sample from you to use
for a cytology test.
➔ Discharge or secretion samples: If you experience
abnormal bodily discharge, such as from your eye, vagina
or nipple, collect a sample of the discharge for a cytology
test.
Fixatives for Cytopathology
95% Ethanol Ideal fixative (recommended) and Yield
100% Methanol enough chromatin detail
80% Propanol Produces less shrinkage more expensive
Denatured
alcohol
Stain
WET FIXED AIR FIXED
Papanicolaou stain Wright stain
H and E staining Giemsa Stain
Mary Grunwald Giemsa Stain
Wright Giemsa Stain
Papanicolaou Stain
1. Hematoxylin: This is a natural dye that stains the cell nuclear
blue. The dye attaches to the sulfate groups of DNA because it has
a high affinity for nuclear chromatins. The most common
hematoxylin dyes used are Harris’ hematoxylin, Gills’ H is the
commonest cytologically although Gills’ hematoxylin and
Hematoxylin S.
2. Orange Green 6: It is an acidic counterstain that stains the
cytoplasm of mature keratinized cells. The components of the
target stain orange in varying intensities of the dye.
3. Eosin Azure: It is the second counterstain, a combination of
eosin Y, light green SF, and Bismarck brown.
➔ Eosin Y stains the cytoplasm of mature squamous cells,
nucleoli, red blood cells, and cilia pink. The eosin dyes
commonly used are EA 31 and EA 50, while EA 65.
➔ Light green SF stains the cytoplasm of active cells such as
columnar cells, parabasal squamous cells, and
intermediate squamous cells, blue.
➔ Bismarck brown Y stains nothing and sometimes it is
often omitted.
Staining dyes will stain different components of the cell with
different colors and intensities as follows:
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 ● Nuclei: Blue ● Prognosis: What is going to happen, curability or non-
● Acidophilic cells: Red curability of the disease?
● Basophilic cells: Blue Green ● Diagnosis: Naming the disease or answer to the
● Erythrocytes: Orange-red to dark pink pathogenesis
● Keratin: Orange-red ● Treatment: What can be done to disease?
● Superficial cells: Pink ● Prevention: How to avoid the complications and spread of
● Intermediate and Parabasal Cells: Blue-Green the disease
● Eosinophil: Orange Red ● BRACHES: Mainly two (2)
● Metaplastic cells: May contain both blue/green and pink ➔ General pathology - dealing with general principles of
● Candida: Red disease
● Trichomonas: Gray-green ➔ Systemic pathology - study of disease pertaining to
specific organ or tissue
Classification for Pap Smear
CLASS PAPANICOLAOU CONCEPTS OTHER PATHOLOGISTS Morphological Branches:
CONCEPTS 1) Histopathology: also known as tissue pathology or anatomic
I Abnormal cells absent Normal or benign pathology.
atypia ● It includes:
II Atypical benign Benign atypia ➔ Surgical pathology - study of removed cell or tissue by
dysplasias biopsy
III Suspicious, including Benign atypia ➔ Experimental pathology - study of disease in
dyskaryosis dysplasias experimental animal
in-situ carcinomas ➔ Forensic pathology - study of organ removed from post-
IV Cancer (fairly conclusive) Benign atypia mortem
dysplasias 2) Cytopathology: study of cells shed off from lesion (Exfoliative
invasive cytology) and fine needle aspiration cytology (FNAC)
few cells ● Hematology: deals with disease of blood it includes
V Cancer (convulsive) In-situ, or invasive with laboratory hematology and clinical hematology
a large number of cells
invasive carcinoma III. CELL INJURY
only ● As the effect of various stresses due to etiological agents a
cell results in change in internal and external environment.
Purpose of Pap Smear ● It is reversible when stress is mild to moderate and
● A procedure in which a small brush is used to gently remove irreversible when it is severe.
cells from the surface of the cervix and the area around it so ● Causes:
they can be checked under a microscope for cervical cancer ➔ Hypoxia (by blood loss)
or cell changes that may lead to cervical cancer. ➔ Physical agents (trauma, radiation etc)
● A Pap smear may also help find other conditions, such as ➔ Chemical agents and drugs
infections or inflammation, or any cellular changes of the ➔ Infectious agents
cervix. ➔ Immunological reactions
➔ Genetic
Infections ➔ Nutritional imbalance
● Fungal Infections – Candida albicans ● Types:
● Bacterial Infection – Gardnerella vaginalis; Decreased level ➔ Reversible (Recovery of cell damage once stress
of Lactobacillus spp. removed)
● Chlamydia – Chlamydia trachomatis ➔ Irreversible (No recovery / Cell death)
● Parasitic Infection – Trichomonas vaginalis
● Cervical Cancer – Human papilloma virus Reversible
● Occurred mainly due to:
II. INTRODUCTION TO PATHOLOGY ➔ Alteration in plasma membrane (i.e. Bleb formation,
Pathos: suffering, logos: study. loosening of intracellular attachment and steatosis:
● Pathology: scientific study of changes (suffering) in the means fat accumulation within cell)
structure and function of the body in disease (impaired ➔ Change in mitochondria (Swelling or hydropic,
health) and it answers the disease in terms of its etiology, rarefaction, phospholipids amorphous densities)
pathogenesis, prognosis and treatment plan. ➔ Nuclear change (Disaggregation of granular and fibrillar
● Lesion: Characteristics changes occurred in cell or tissue as element)
the result of disease. ➔ ER changes (Dilatation, detachment and disaggregation)
● Etiology: Causative factor
● Pathogenesis: Mechanism by which lesion or disease Irreversible
produced ● Mainly occurred as the result of:
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 ➔ Swelling of ER histological (E.g TB, Leprosy and syphilis)
Specific
➔ Lysosomes rupture feature
➔ Nuclear condensation According to Aseptic due to sterile chemical agents
➔ Nuclear lysis causative due to pathogens
➔ Membrane blebs Aseptic
factors
➔ Swollen mitochondria with amorphous densities
● The irreversible is mainly of two types: Necrosis and Cardinal Feature
Apoptosis ● Redness: Due to excess flow of blood
➔ Necrosis: It is death of a localized area of tissue followed ● Swelling: Due to accumulation of intravascular contents to
later by degradation of tissue by lysosomal enzymes that interstitial space
mainly occur in inflammation and in hypoxia. ● Heat: Due to change in thermoregulation as the result of
➔ Apoptosis: Also known as coordinated and programmed injury
death of cell occurred mainly in pathological condition ● Pain: due to peripheral nerve irritation by chemicals
but not in inflammation secreted on injury

IV. CELLULAR ADAPTATION A. Shock

● Form of Reversible cell injury
● Cell or cellular adaptation: capability of adjusting their
structure and functions in response to various physiological
and pathological stimuli (Mild to Moderate)
● Types:
➔ Physiological: Occurs in response to an stimulus and
ceases once the need of adaptation has ceased e.g.
change in breast and uterus during pregnancy due to
influence of hormones
➔ Pathological: Occurs in response to injury or pathogens
by producing cell stress proteins, which protect from
damage and help in recovery
● Atrophy: Reduction of cell size which results in shrinkage of
cell or organ. Due to degradation of cell protein by
lysosomal enzymes e.g. ischemic atrophy, disuse atrophy of
muscle
● Hypertrophy: Increase in the size of cells which result in
enlargement of cell or organ. Due to increased production of
cellular protein, intracellular factor and growth factor e.g.
Left ventricular hypertrophy
● Hyperplasia: Increased number of cells in an organ or tissue.
Due to synthesis of DNA and Proliferation of cell by local
production of growth factor e.g. Hyperplasia of
endometrium and hyperplasia of prostate
● Metaplasia: Replacement of one adult cell type with
another. Due to long time persist of cause leads to
Metaplasia by the influence of different factors like growth,
cytokines etc. e.g. Squamous metaplasia, columnar
metaplasia
Note: Both Hyperplasia and Metaplasia are fertile soil for the
development of malignancy in future if cause or stimuli is not
removed
V. INFLAMMATION
CRITERIA TYPES DEFINITION
Lasting for seconds, minutes or
Acute
According to <48hrs
duration Subacute or Lasting >48hrs, days, weeks,
Chronic months or years
According to Non specific (Majority of cases)
● also known as cardiovascular collapse, as the result of
reduced circulating blood volume and or inadequate
perfusion of cells and tissue.
● Pathogenesis: All forms of shock involve:
➔ Reduced effective circulatory volume
➔ Impaired tissue oxygenation
➔ Release of inflammatory mediators.
● Types:
➔ Hypovolemic shock: due to acute hemorrhage, Burns,
Excessive use diuretics
➔ Cardiogenic shock: due to MI, Cardiac arrhythmias,
pulmonary embolism
➔ Septic Shock: Due to Gram negative and positive
bacterial infection
➔ Other shock: includes traumatic shock due to severe
injury, neurogenic shock due to head injury
● Clinical features:
➔ Low blood pressure
➔ Low body temperature
➔ Feeble pulse
➔ Pale face
➔ Shallow respiration
➔ Cold clammy skin
● Stages: Reversible and irreversible stage of shock
B. Hemorrhage
● Escape of blood from the rupture or non-ruptured blood
vessel
● Types:
➔ Internal (bleeding within body) and external (bleeding
out of body) hemorrhage
➔ Acute (sudden and massive bleeding) and chronic (small
amount over a period)
Classification of hemorrhage
● Class I Hemorrhage involves up to 15% of blood volume.
There is typically no change in vital signs.
● Class II Hemorrhage involves 15-30% of total blood volume
tachycardia, difference between the systolic and diastolic
blood pressures, peripheral vasoconstriction, pale and cool
skin. Blood transfusion is not usually required.
● Class III Hemorrhage involves loss of 30-40% of circulating
blood volume, blood pressure drops, the heart rate
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 increases, shock, diminished capillary refill, blood ● It is the process of tissue death (necrosis) as the result of
transfusion are usually necessary. ischemia
● Class IV Hemorrhage involves loss of >40% of circulating ● Causes:
blood volume, aggressive resuscitation is required to ➔ Occlusion (due to thrombus or embolus)
prevent death. ➔ Trauma
➔ Others (Atherosclerosis, hypoglycemia etc)
● Causes: ● Pathogenesis: As the result of injury or ischemia there will
➔ Trauma to the vessel be slowly death of the cell or tissue as the result of changes
➔ inflammation of vessel wall in vascular and cellular level like irreversible form of cell
➔ vascular diseases like atherosclerosis injury
➔ aneurysm etc ● Types:
➔ elevated pressure within vessel ➔ Anemic and hemorrhagic infarction
➔ low platelet count ➔ Recent and old infarction
➔ hemophilia etc
● Effect: G. Neoplasm (Tumor)
➔ effect of blood loss depends on the amount ● Neo: new and plasia: growth, so neoplasia means abnormal
➔ speed and site of blood loss growth of tissue or organ.
➔ Up to 33% of sudden blood is fatal may cause death ● It is also known as tumor, the branch which deals about
neoplasm is oncology.
C. Thrombosis (Clot) ● Types:
● Process of formation of clot mass in circulation from the ➔ Benign (Non- Cancer, Non harmful)
constituents of blood, the mass is called as clot or thrombus ➔ Malignant (Cancer, Harmful)
● Pathophysiology:
➔ Epithelial injury Difference between Benign and Malignant Tumor
➔ Plate abnormality FEATURES MALIGNANT BENIGN
➔ Alteration of blood flow Metastasis Present Absent
➔ Abnormality of coagulation system Size Usually large Usually small
Surrounding invade Compressed
D. Embolism tissue
● It is the process of partial or complete obstruction of some Spread Spread to other parts Non-spread
part of the cardiovascular system by mass (embolus) carried Differentiation Undifferentiated Well
in circulation. differentiated
● This embolus may be a blood originated (thrombus) or fat Treatment Chemotherapy, Surgery, Radiation
(fat embolus) or air (air embolus) etc, Radiation
● Types:
➔ Venous, arterial, paradoxical (venous to artery) Cancer Grade
➔ Solid, liquid and Gaseous embolism A cancer’s grade describes how abnormal the cancer cells and
➔ Bland (when embolus is sterile) and Septic ( infected tissue look under a microscope when compared to healthy cells.
embolus) ● Grade 1: Tumor cells generally resemble healthy cells (well-
differentiated).
E. Ischemia ● Grade 2: Tumor cells are somewhat abnormal (moderately
● It is deficient blood supply to a part of tissue relative to its differentiated).
metabolic needs ● Grade 3: Tumor cells are very abnormal (poorly
● Types: Complete ischemia and Partial Ischemia differentiated).
● Pathophysiology: ● Grade 4: Tumor cells considered the most abnormal
➔ Ischemia either due to Hypoxia (low oxygen, low (undifferentiated).
hemoglobin, or low blood supply)
➔ Mall nourishment of cell and inadequate clearance of While a grade describes the appearance of cancer cells and tissue,
metabolites a cancer’s stage explains how large the primary tumor is and how
● Causes: far the cancer has spread in the patient’s body.
➔ Occlusion (due to thrombus or embolus) ● Stage 0: The cancer is in-situ or found where it started and
➔ Trauma before it has spread anywhere.
➔ Others (Atherosclerosis, hypoglycemia etc) ● Stage 1: The cancer usually hasn’t grown into nearby tissue
and hasn’t spread to the lymph nodes or other parts of the
body.
● Stage 2: The tumor has grown and cancer cells may be found
in nearby lymph nodes.
F. Infarction

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 ● Stage 3: The cancer is larger or embedded more deeply into
nearby tissue or may have formed multiple tumors. It may
have spread to nearby lymph nodes.
● Stage 4: The cancer has metastasized and spread to other
organs or distant parts of the body.
Gleason grading system is used to help evaluate the prognosis of
men with prostate cancer using samples from a prostate biopsy.
FIGO (International Federation of Gynecology and Obstetrics) for
Gynecologic cancer
● These changes occur along a continuum and can be helpful
in determining the post-mortem interval, which is the time
between death and examination.
The stages that follow shortly after death are:
● Corneal opacity or "clouding"
● Pallor mortis, paleness which happens in the first 15–120
minutes after death. Meaning “paleness of death” - happens
a few minutes after passing simply because the heart stops
pumping.
● Algor mortis, the reduction in body temperature following
death. This is generally a steady decline until matching
ambient temperature. coldness of death” happens relatively
quickly, since the heart is no longer pumping warm blood
throughout the body. Roughly 1 hour post-mortem.
● Rigor mortis, the limbs of the corpse become stiff (Latin
rigor) and difficult to move or manipulate. During the
“stiffness of death,” the body produces a copious amount of
lactic acid. 2 to 48 hours post-mortem.
● Livor mortis, or dependent lividity, a settling of the blood in
the lower (dependent) portion of the body. “bluish color of
death” 20 minutes to 12 hours post-mortem.
● Putrefaction, the beginning signs of decomposition

The cardinal signs of death may refer to the ending of breathing,

heartbeat and circulation, or to algor mortis, livor mortis and rigor
mortis; the adoption of brain death as a definition has lessened
the centrality of these signs.
In a clearer contemporary terminology, algor mortis, livor mortis
and rigor mortis are called "early postmortem" changes, in
distinction from the "immediate postmortem" changes associated
with the cessation of bodily functions, as indicated by vital signs.

Those stages are followed, in taphonomy, by

● Decomposition: the reduction into simpler forms of matter,
STAGE FINDINGS accompanied by a strong, unpleasant odor.
IA Micro-invasive carcinoma. ● Skeletonization: the end of decomposition, where all soft
IB Macroscopic Invasive cancer confined tissues
to have decomposed, leaving only the skeleton.
the cervix. ● Fossilization: the natural preservation of the skeletal
IIA remains formed over a very long period. This stage may not
Tumour extending to upper third vagina
but not to the parametrium occur, depending on the circumstances and the conditions
IIB Tumour extending to the parametrium of the surrounding environment.
IIIA Tumour involving the lower third vagina
with no extension to the pelvic side wall VII. AUTOPSY
IIIB ●
Tumour extending to pelvic side wall Autopsy pathology is the practice of medicine that directs its
and/or hydronephrosis or non- efforts to the scientific study of the human body after death.
functioning kidney ● The objectives of the autopsy include:
IV A ➔ the evaluation of clinical diagnoses
Tumour involving adjacent pelvic organs
i.e. bladder or rectum. ➔ the detection and diagnosis of unsuspected diseases
IV B Extra-pelvic spread, e.g. metastasis➔ the study of the cause, nature, and development of
to the
liver, lungs etc disease
➔ determination of the cause of death
VI. POST MORTEM ➔ assisting in the assessment of the validity, value, and
appropriateness of diagnostic and therapeutic
● Post-mortem changes refer to the series of changes that
procedures
occur to a body after death.
➔ provision of information to families, physicians, and
● These changes can generally be divided between early post-
society
mortem changes and late post-mortem changes (also known
➔ quality assurance in medicine
as decomposition).
➔ medical education and training

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Three different types of incisions conventionally used for opening
the neck, thorax and abdomen are:
1. “I” Shaped Incision: It starts from symphysis menti and extends
straight to symphysis pubis right or left to umbilicus.
● Disadvantages: The prominent stitch mark in front of neck,
thorax, abdomen and also the neck structures especially in
the back of neck are not visualized.
2. “Y” Shaped Incision: Starts at a point close to the acromion
process extending downwards below the breast and across to the
xiphoid process in both sides, then from the xiphoid process, the
incision is extended downwards to the symphysis pubis. Here,
visualization of neck structure is very poor but in this incision the
stitch mark in front of the neck is absent.
3. Modified “Y” Shaped Incision: Starts behind the user to
midclavicular point bilaterally, then carried out over the clavicle to
suprasternal notch and then a straight incision to symphysis pubis
in midline.
● Disadvantage: The prominent stitch mark in front of neck
and poor visualization of back of neck though exposure of
the neck structure in front of neck and to some extent the
side of neck is better.
4. Cosmetic Autopsy Incision: using this incision we can expose
both the front as well as the posterior aspect of the body and at
the same time hide the stitch marks in the front of the body
Autopsy Techniques
A. Virchow Technique
● The organs are removed one by one and dissected as
removed.
● This approach is good for demonstrating pathological
change in individual organs, especially in high risk autopsies
or where permission is limited to one organ.
● This organ can be immediately removed and examined.
● The disadvantage of this technique is that relationships
between various organs may be hard to interpret.
B. Rokitansky Technique
● This procedure is characterized by in situ dissection, in part
combined with en bloc removal.
● The term “Rokitansky technique” is used erroneously by
many pathologists to designate the en masse technique.
C. En Masse Technique
● Thoracic, cervical, abdominal, and pelvic organs are
removed en masse and subsequently dissected into organ
blocks.
● This is the best technique for preserving the vascular supply
and relationships between organs.
● Another advantage is that the body can be made available to
the undertaker quickly, without having to rush the dissection
and risk obscuring findings or destroying important
specimens.
● The major disadvantage is that the organ mass is often
awkward to handle, and the autopsy is difficult to perform
without an assistant.
D. En Bloc Technique
● Various modifications of the en bloc technique are widely
used.
● Thoracic and cervical organs, abdominal organs, and the
urogenital system are removed in functionally related
blocks.
● This procedure is a compromise between the Virchow and
en masse techniques, preserving anatomical relationships
sufficiently for most cases while being simpler for one
person to execute.