Non-gynaecological

cytology
Prepared by: Ms. Roselin Tsauses
Anatomical Pathology 2B (ANP621S)
September 2021
Recall from previous lectures…

• Cytology can be performed on any type of tissue specimen to look

for neoplastic cells.
• Neoplasia, which literally means “new growth,” is the process of excessive
and uncontrolled cell proliferation (Kumar et al., 2010).
• This refers to an abnormal mass of tissue that forms when cells grow and
divide more than they should or do not die when they should.
• Neoplasms may be benign (not cancer) or malignant (cancer).
• Benign neoplasms may grow large but do not spread into, or invade,
nearby tissues or other parts of the body.
Contents
• Type of specimens for NGC
• Fine needle aspiration (FNA) cytology:
• Principle, technique, equipment, preparation,
precautions, advantages, disadvantages, causes of
error
• Lesions: Palpable & Non-palpable
Type of specimens for NGC

• Cytology of urine, anatomy of UT & specimen types.

• Serous effusions & peritoneal washings, anatomy of pleura,
peritoneum, pericardium, specimen types.
• Lower respiratory tract cytology, anatomy & specimen collection.
• Fine needle aspiration cytology
Cytology of urine

Objectives:
• Describe the normal anatomy of the urinary tract (UT).
• Describe the histology of urothelium.
• List common specimen types and preparatory methods.
• Describe cytological constituents of voided urine.
• Explain aetiology, symptoms and treatment of bladder cancer.
Anatomy of the UT
Urothelium

• The renal pelvis, the ureters, the bladder, and parts of the urethra
are lined by specialized type of epithelium known as urothelium.
• Small portion of the urethra are lined by squamous epithelium.
• Urothelium was originally called transitional epithelium as it was
thought to be a transition between squamous and glandular
epithelium. This term is still in use.
• Urothelium is impermeable to urine and does not allow the
noxious substances in urine to cross back into the blood.
• Other feature making this epithelium unique is that it has
specialized structures within, which allows the urothelium to
expand and contract depending on the volume of urine in the
bladder.
Urothelium…
• This is a multilayered epithelium:
• In some respects, it is similar to the cervical epithelium previously learned
as part of the FGT.
• Layers of the squamous epithelium:
• Basal, parabasal, intermediate, squamous
• Compared to cervical cytology, these cell layers are not easily identified
in urothelium and most cytologists refer to them as deeper layer cells
and superficial cells.
• Deeper layer cells are similar to parabasal squamous cells and have
dense staining cytoplasm with sharp border.
• The superficial layer cells are fairly large cells, and may have a single
nucleus, two nuclei (binucleated), or multinucleated and cover
intermediate cells underneath.
• For this reason, you may hear them being referred to as umbrella cells.
Urothelium…

• Unlike squamous and other epithelia, the urothelium is a very low

turnover/ exfoliation epithelium that takes almost a year to shed.
• In common with other epithelia, the urothelium can also undergo
the process of metaplasia.
• Common forms of metaplasia in the UT include: squamous and
glandular metaplasia.
Histology of urothelium
Histology…
Histology…
UT: Specimen types
Three main type of urine specimens:
• Voided:
• Most common sample type
• Patient produces a sample in a pot
• Samples undergo degeneration very quickly, therefore the pot must have a preservative.
• Preervatives reduce cellular degeneration and stop bacterial growth.
• Ileal:
• Useful for follow-up of patients who have been treated for bladder cancer, as they have an
increased risk of developing cancers in the ureters for the kidneys.
• An ileal conduit is a “urinary diversion” that is constructed from a section of ileum when a patient
has had their bladder removed during surgery for bladder cancer.
• Urine samples from these patients have a varied appearance:
• May show large numbers of degenerating intestinal epithelial cells, macrophages, inflammatory
cells and mucus.
• Easy to over-look occasional malignant cells due to busy background.
• Catheter:
• Urinary catheter is a short piece of thin plastic tubing that is inserted into the bladder to allow
urine to drain freely into a bag.
• Urine from catheter specimens is often collected from a bag which has been at room temp. for
several hrs, so cells may show degenerative changes.
Serous effusions & peritoneal washings
Objectives:
• Describe the anatomy and physiology of serous cavities.
• Describe different types of serous effusions and factors relating to
their formation.
Anatomy & physiology (A&P) of
serous membranes
There are three body cavities:
• Pleural
• Peritoneal
• Pericardial
• The above cavities are lined by a membrane known as the
mesothelium.
• Cells forming the mesothelium are called mesothelial cells.
• Immediately below these cells is a thin layer of fibrous connective
tissue, which includes lymphatic vessels and capillaries.
• Mesothelium is composed of two layers:
• Parietal layer: covers the wall of the cavity
• Visceral layer: lines the surface of the organs
• The mesothelium produces serous fluid, a lubricating fluid, allowing
movement of organs in body cavity e.g. heart beating, bowel
movement, expansion and contraction of lungs.
A&P of serous membrane…

• Different names of mesothelium according to site:

• Pleural mesothelium: covers lungs
• Peritoneal mesothelium: lines most abdominal organs
• Pericardium: surrounds heart
• Mesothelium also covers male and female reproductive organs.
• Males: Tunica vaginalis testis covers testicles
• Females: Tunica serosa uteri covers parts of uterus.
A&P of serous membrane…

• Imbalance in the:
• hydrostatic pressure within capillary lumen ,
• colloid oncotic pressure (osmotic pressure caused by proteins in plasma),
• rate of lymphatic drainage and
• permeability of capillaries result in accummulation of fluid termed as:
• Effusion: classified according to amount of protein they contain as:
• exudates: Higher protein and high specific gravity.
• Caused by increased permeability of capillaries when serous membranes are
damaged by a disease process. Metaplastic malignancy and infections are common
causes of exudates.
• transudates – have low protein and low specific gravity (density), low cellular content
• Caused by imbalance in hydrostatic and oncotic pressure – associated with kidneys,
heart, liver failure or hypoalbuminaemia.
• Ascites
Pericardium
Pleura
Peritoneum
Specimen collection and sample
processing
• Aspiration of serous fluids is usually done under ultrasound
guidance using local anasthesia.
• It involves the insertion of a needle or cannula (tube) into the
serous cavity, procedure termed as:
• Thoracocentesis in the pleural space
• Pericardiocentesis in the pericardial space
• Paracentesis in the peritoneal cavity
• The fluid is often collected in a bag or large syringe.
• For cytological analysis at least 20mL of fluid is needed; optimally
40mL will provide additional material for ancillary techniques.
• Specimen should be sent to the lab without delay.
• Some serous effusions are rich in fibrin (protein involved in
clotting of blood), this may cause the sample to clot on standing.
Specimen collection

• Clotted samples cannot be processed using cytological preparation

techniques, but can be fixed in formaldehyde and processed as a histological
specimen.
• To prevent fluids from clotting, anticoagulants may be added to the sample.
• Sodium citrate is a suitable cytological anticoagulant.
• Sample preparation:
• Two alcohol fixed slides are prepared for Pap stain – allows optimal assessment of nuclear details.
• Two air-dried slides for Diff Quick stain (Romanowsky type stain) – better for visualizing
cytoplasmic features.
• Prior to processing, observe macroscopic appearance and volume of sample
– helps to relate cytological picture to macroscopic appearance of the fluid.
• For samples that do not appear to contain blood, the cytocentrifuge is used.
• For blood filled samples, density gradient method is employed.
A&P of Lower respiratory tract (RT)
cytology
Objectives:
• Describe the anatomy of the respiratory tract.
• Describe the normal cells found in respiratory samples.
• Outline different sampling and preparation techniques.
A&P of upper & lower RT
Normal cells found in respiratory samples
Two main types of epithelium line the RT:
• Non-keratinizing squamous epithelium:
• Covers areas that are liable to frictional forces e.g., caused by swallowing of food or by touch.
Includes: front part of nasal cavity, central and lower portions of pharynx and parts of larynx.
• These type of epithelial cells commonly exfoliate.
• They can be recognized in respiratory samples as: superficial and intermediate squamous cells –
similar in all aspects to those in the FGT that you have learned.
• Respiratory epithelium (type of columnar epithelium):
• Lines major part of nasal cavity, parts of larynx, trachea and bronchi.
• These type of epithelial cells are referred to as pseudostratified epithelium, because it appears to
be multilayered, even though it consists of a single layer of epithelial cells.
• Impression of multi-layering is due to the position of nuclei in different levels of cells in the
epithelium.
• Respiratory epithelium consists of ciliated columnar cells interspersed with mucus secreting goblet
cells.
• Ciliated cells have basal, round to oval shaped nuclei with finely granular chromatin, with the
luminal part (facing the lumen) covered by cilia; these are numerous and at their point of
attachment form a terminal plate.
Respiratory epithelium (RE)…

• Other cells found in the RE identified easily in cytological samples:

• Basal cells or reserve cells (precursor of goblet and columnar cells)
• Neuroendocrine cells
• Non-ciliated columnar cells called Clara cells (lines the terminal bronchiole).
• Cells lining the alveoli are called:
• Type I pneumocytes: large, thin cells stretched across a large surface and cover the areas where
gaseous exchange takes place.
• Type II pneumocytes: produce surfactant, which has a dual function of preventing alveoli colapse
during the breathing cycle and protecting the lungs from injuries and infections caused by foreign
bodies and pathogens.
• Also act as reserve cells or progenitor cells and can differentiate into type I pneumocytes
when they need to be replaced.
Pseudostratified epithelium
Respiratory epithelium (RE)…

• Alveolar macrophages (also known as histiocytes) are:

• Macrophages found in the pulmonary alveolus
• They are phagocytes: role of engulfing, digesting and removing inhaled particles and pathogens.
• Stimulate lymphocytes and other immune cells to respond to foreign matter.
• Cytoplasm may be vacuolated with evidence of inhaled particles (usually carbon) may be seen as
brown granules on Pap stain or black in Romanowsky stains.
• May have single round or bean-shaped nucleus, with granular chromatin.
• Bi-nucleation and multi-nucleation may occur, particularly in response to foreign matter.
Alveolar macrophages
Other cellular & non-cellular
components of the RT
Curschmann’s spirals (named after German physician Heinrich
Curshmann 1846-1910) who first described them as:
• Strands of mucus that are formed in the lumen of small bronchi.
• Occassionally seen in RT samples.
• It is thought that their presence was diagnostic of conditions such as asthma (we now know that they
can be seen in any condition where there is an increase in mucus production.
• Vary in structure, but have coiled appearance, with dark central part and translucent periphery.
• Inspissated mucus refers to thickened mucus, looks different to
Curschmann’s spiral in that it has no structure and stains dark blue
in Pap stain.
Other cellular…

• Charcot-Leyden crystals (1st described by French physician Jean-

Martin Charcot 1825-1893 and German physician Ernst Viktor von
Leyden 1832-1910):
• Orangeophilic, needle-shaped structures derived from degenerating eosinophils.
• Seen in patients with allergic disorders such as asthma or eosinophilic pneumonia.
• Ferruginous (containing particles of iron) or asbestos bodies are
formed when filamentous particles become coated in protein and
iron.
• This is often due to inhalation of asbestos, where small asbestos fibers are enveloped by
macrophages.
• Often called asbestos bodies.
• Undigested food particles:
• E.g. Fragments of plant tissue and meat fibres are sometimes observed in sputum samples.
• Plant tissue have characteristic rectangular shape.
• Meat fibres can be recognized by presence of cytoplasmic cross striation.
Curschmann’s spirals
Asbestos bodies
 Charcot-Leyden crystal

Charcot-Leyden crystal in sputum

smear
Specimen collection and sample
processing
• Sputum:
• Mixture of mucus cells that is expectorated (coughed up) from respiratory tract.
• Patient collects sputum from early morning deep cough into specimen jar.
• Bronchial brushings:
• Taken from surface of a tumour visualized with the bronchoscope.
• Brush gently rolled onto the surface of the glass slide and air-dried for Diff-Quick staining and
rinsed in LBC preservative to allow production of further slides and cell blocks for
Immunocytochemistry (ICC).
• Bronchial washings:
• Involves instilling several 20mL aliquots of isotonic saline through a bronchoscope and with the aid
of suction aspirating the material into a trap.
• The fluid will include mucus and cells which have been exfoliated. The fluid is centrifuged and the
concebtrate is used to make smears, or is processed using LBC methodology.
Bronchoscopy
Fine needle aspiration
• Fine-needle aspiration (FNA) biopsy is a reliable, cost-effective
procedure that may be useful in the workup of patients with both
palpable and deep-seated mass lesions.
• An extremely simple procedure technically, FNA biopsy involves
the introduction of a small-gauge needle into the mass and the
extraction of representative cellular material.
• Numerous large series studying the diagnostic accuracy of this
procedure in a variety of organ systems (including the
gynecologic tract) have repeatedly confirmed it as both a
sensitive and specific test that may be performed with minimal
morbidity and at a relatively low financial cost to the patient.
• Source: McGrath, C, Yu, G, et al, Glob. libr. women's med.,
(ISSN: 1756-2228) 2008; DOI 10.3843/GLOWM.10264
FNA
• Purpose – obtain diagnostic material for cytologic study from
organs that do not shed cells spontaneously.
• Organs may include:
• Lungs
• Liver
• Breasts
• Thyroid
• Lymph nodes
• Ovaries
• Testes, etc.
Principle: FNA
• Based on negative pressure
• Needle is positioned within the target tissue and negative pressure is applied by
pulling the plunger.
• Needle is then moved back and forth within the target tissue.
• The function of negative pressure is not to tear cells from the tissue, but to hold the
tissue
against the sharp cutting edge of the needle.
• Softer tissue components, e. g. tumor cells protrude over the edge, are cut or
scraped off and accumulate in the lumen as the needle advances through the tissue.
• To obtain the greatest cellular yield, the needle is moved back and forth within the
lesion
along the same rack with negative pressure maintained.
• The negative pressure is released while the needle remains in the target tissue.
• The needle is withdrawn from the tissue and then detached.
• Air is then drawn into the syringe and the aspirated material will flow into the syringe.
FNA: Technique
• The syringe used has a special handle which allows a single-
hand grip while the biopsy is being made.
• The inner surface of the barrel is coated with silicone to
ensure ai tight fitting of the plunger during aspiration.
• The length of the needle varies according to the site of biopsy.
• Standard needles for soft tissue puncture have an outer
diameter of 0,5 – 0,9 mm.
• Thicket needles with an obturator for obtaining material
from sclerotic/ bony tissues have a diameter of 1 – 2
mm.
FNA: Technique…
• Fine needles are usually used and if they are not
satisfactory; a thicker needle with a mandrin may be
used.
• The needle may be introduced by a slow rotary movement
under moderate pressure.
• When the lesion is reached, the mandrin is withdrawn and the
syringe is attached to the needle.
• Vigorous suction takes place for aspirating the cells from the
lesion.
• The aspirate is placed in a watch glass and large tissue
fragments are sent to histology, while small fragments are
deposited on a glass slide.
 FNA -
Syringe
Aspiration techniques
• For successful FNA cytopathology three criteria must be
met:
• Aspiration of adequate and representative material
• Correct processing of specimen.
• Informed interpretation and the issue of an accurate
report.
Equipment
• Basic tools required consists of a needle and syringe.
• Needles:
• To reduce bleeding, it is important to use as fine a needle as possible.
• Length of needle varies according to the site of the biopsy.
• Standard needles for soft tissue puncture have an outer diameter of
0.5 – 0.9 mm.
• Thicker needles with an obturator for obtaining material from sclerotic
or
bony tissues have a diameter of 1 – 2mm.
• For deeper lesions, longer specialist needles are available.
Equipment…
• Syringes and holders:
• Standard 10- and 20-ml disposable syringes produce sufficient
negative pressure.
• Many aspirators prefer to use 10ml syringes as the small size
makes them easier to handle.
• A syringe holder may be used when aspirating palpable lumps as
they allow for one-handed operation; the free hand may be used to
immobilize the lump.
• Glass slides:
• Frosted end slides are suitable for immediate labelling with a pencil
and can be used as an implement for making direct preparation.
Equipment…
• Fixative and transport media and routine staining methods:
• Direct preparations can be spray fixed for Papanicolaou staining or
immersed in 95% alcohol, but if on-site assessment is available, it is
recommended to make a maximum of two air-dried direct smears and
stain these with Romanowsky stain such as Diff-Quick and check for
adequacy.
• The content of the needle and subsequent samples should be
rinsed in transport media for processing in the laboratory.
• There are different transport media available. Some have been
specifically designed for use in cytology: these include commercial
LBC preservatives such as CytoLyt or CytoRich.
• The success of FNA depends on accuracy and specificity, which can
be greatly enhanced by high quality preparations and by the
application of ancillary techniques.
Precautions
• FNA technique must be performed by a trained experienced
clinician.
• Speed and gentleness are essential prerequisites for the
preparation of high quality slides.
• The lump must be palpated and its boundaries must be
defined before the needle is inserted.
• Always maintain negative pressure while aspirating.
• Make sure that nothing but air is within the barrel of the syringe
and that all aspirated material is within the lumen of the
needle before withdrawing from the lesion.
• The needle or syringe must be washed with sterile saline.
Precautions…
• Excessive blood in the preparation must be avoided; this
causes dilution of the cells.
• Patient slides must be labelled correctly.
• Do not spatter cellular material onto the slides, this will
cause significant drying artefacts.
• Instead, prepare a thin, evenly distributed smear of the
aspirated material.
• Fix all smears immediately to prevent cellular distortion.
Preparation of smears
• The cellular material is in the needle.
• Without aspirating air, expel the contents onto a glass slide.
• Disconnect the needle and aspirate air into the syringe.
• Reconnect the needle to the syringe. Now expel the content in the
needle again. Repeat about 3 times in total.
• You must work quickly because the material must not dry out.
• Gently place a second slide over the aspirated material on the
glass slide and wait briefly for the material to spread by capillary
action.
• Then gently slide the two slides apart -- do not press -- this will
damage the cells.
Preparation of smears…
• Air dry the top slide -- for Giemsa stains.
• Immediately spray fix the bottom slide with cytological spray
fixative, at a distance of 25cm, gently and liberally.
• Do NOT air dry the bottom slide, this is where most of the cellular material
is, and it is needed for routine PAP stains.
• Repeat the whole process with a new needle and syringe. Perform 2-3
passes per lesion (and end up with 4-6 slides).
• Label the slides with a hospital or file number.
• Wait for the slides to dry completely.
• Wrap in cardboard or plastic slide trays, or in paper -- to protect it
against breakage.
• Fill in a request form with relevant history. Send to the laboratory --
Cytology section for examination by a cytopathologist/
cytotechnologist.
Advantages
• Safe.
• Quick investigation with rapid report.
• Highly sensitive and specific for the diagnosis of malignancy.
• Causes minimal inconvenience to patient.
• Outpatient or bedside procedure.
• Cuts down on bed occupancy for diagnostic investigations.
• Allows pre-operative diagnosis, informed patient consent and planned
surgery.
• Prevents in many cases, the necessity of frozen sections.
• Allows definitive diagnosis on inoperable patients.
• No problems with wound healing.
• Readily repeatable.
• Highly cost-effective.
Disadvantages
• Practice and skill in aspiration techniques are
necessary.
• A percentage of aspirate is unsatisfactory.
• Experience is required for accurate interpretation.
• Diagnostic information is limited.
Causes of error (Danger of FNA)
• Excessive necrosis or inflammation could lead to a false
negative report.
• Poorly preserved or poorly stained material could lead to an
incorrect diagnosis.
• Specimen not representative of the lesion i.e. the mass was
missed during aspiration – this could result in a false
negative report.
• Dangers associated with the procedure itself e. g.
haemorrhage, pneumothorax, spread of tumour.
Lesions: Palpable & non-palpable

Main difference between palpable lesions and non-

palpable lesions.
• Palpable lesions: can be touched or felt easily.
• Non-palpable lesions: represent a challenge in obtaining a
sample for cytological examination. Lesions of visceral
organs such as bones.
 No. Palpable Non-palpable lesions
lesions
1. Breast Lungs
2. Lymph notes Liver
3. Thyroid Pancreas
Palpable 4. Salivary glands Kidneys, adrenals,
and non- retroperitoneum, and gonads,
palpable i.e.,
lesions
ovaries, testes
5. Prostate Bone
6. Skin and soft Orbit and eye globe
tissues
7. Central Nervous System
8. Pleura and mediastinum
Test your knowledge
1. You are requested to screen cytological specimens from the respiratory
tract. Name and briefly describe cells and elements you may find in
these specimens.
2. Briefly explain how you would obtain diagnostic material for cytologic
study from organs that do not shed cells spontaneously by:
(a)Referring to the principle behind the relevant technique.
(b)Identifying and briefly describing the equipment required for this
technique, supported by a relevant illustration of the item used to perform
the technique explained in question 2 (a).
3. Once you have obtained the diagnostic material for cytologic study as
explained under question 2 (a), further discuss how you would go about
preparing smears before it is sent off to the Cytopathologist/ Cytotechnologist
for examination.
4. Explain the main difference between palpable lesions and non-palpable
lesions.
 THE END.
THANK YOU!