Diagnostic cytology

Kagira JM
• DIAGNOSTIC CYTOLOGY = Exfoliative Cytology;
Cytopathology
• Defn: examination of individual cell details for the purpose of
diagnosis & prognosis
• Advantages over histopathology:
a. Samples are cheaper & easier to obtain
b. Samples are simpler to process & result quickly obtained
c. Agents of disease easier to detect - resolution at 100 x mag
• Cytological preparations are stained with stains, e.g. Giemsa
• Histopath examination supportive and gives tissue
architecture

sputum
Bacteria in sputum
A) SOLID TISSUE CYTOLOGY
i) Swabs
• Used as a collection method for sites such as body cavities or fistulous
tracts eg.:
– Nasal cavity (i.e., looking for fungal spores)
– Vagina (i.e., establishing which phase of the estrous cycle)
• Goal - collect superficial cells or bacteria for preliminary or diagnostic
cytology or culture
• Cells are delicate in nature, and a gentle approach should be taken during
collection
• Insert the swab into the desired cavity and gently roll the swab against
the mucosal surface or lining of the fistula, collecting the superficial cells
• Transfer the collected cells onto microscope slides, gently roll the swab
along the length of the slide
• Allow slide to air dry and stain.
A) SOLID TISSUE CYTOLOGY
(ii) Tissue imprints/scrapings are useful for:
• Examining accessible soft tissues (biopsy & necropsy
material)
• Surface blotted free of debris & a slide touched onto the
exposed surface
• Examine: individual cell details, cell relationships &
etiological agents
• Tissue scrapings also done (eg., conjunctiva) – impression
smear then done
(iii) Fine needle cell aspirates:
• In living animal (internal & external solid/fluid masses)
• Cellular material aspirated with fine gauge needle & syringe
• Placed on a slide and gently smeared
(iv) Tissue biopsy
• Involves sampling a small section of tissue, rather than individual cells
• Sample is useful for cytological or histological examination
• Sites biopsied = superficial locations, eg., skin or masses, internal organs
and intestinal biopsies
• Wedge biopsies
– Collected using a scalpel blade to excise a small piece of tissue
– Sample contains tissue from the lesion, the transition zone, and
normal tissue
• Biopsy punch
– Useful for collecting small, round biopsy specimens
– Punch has a circular blade that cuts easily with gentle rotation
Examination of solid tissue cytology
– As for histopathology: detect, describe & deduce
– Cellular detail, acellular, & agents of disease detected
– Lesions can be:
1. Proliferative (hyperplastic or neoplastic): benign or malignant
2. Inflammatory or degenerative (acute and chronic) processes
– Detecting agents of disease:
1. Physical (e.g. foreign bodies),
2. Living (e.g. acid fast bacteria, toxoplasmal organisms, chlamydial
organisms)
3. Chemical (e.g. calcification of tissue)
• Eg: lymphadenomegaly
– Fine needle cell aspirate: lymphoid hyperplasia,
lymphadenitis, lymphosarcoma & Theileria parva parva
1. Primary degenerative lesions
• Lesions cystic in nature (e.g. epidermal cyst)
• Deposits of minerals or pigments (e.g.haemosiderin)
• Physical damage (e.g. physical trauma or toxic
substances)

2. Inflammatory lesions
• Presence of inflammatory cells or by the presence of
infectious agents (e.g. bacteria, viruses, fungi)
• Detection of Distemper virus or chlamydial inclusions
in conjunctival smears
• Inflammatory lesions: characterised by agents & type
of cells
• Neutrophils:
– Acute inflammation due to infective agents such as bacteria
– Chronic inflammation if the chemotactic factor (such as
bacteria) is persistent
– Purulent exudate refers to a neutrophil predominance
• Eosinophils :
– Response to some parasites, allergies
– Lesions involving mast cells (e.g. skin, respiratory tract)
• Macrophages (histiocytes) :
– For phagocytosis of macromolecular material
– Found in chronic inflammatory lesions
– In tissue damage or foreign debris
• Lymphocytes & plasma cells:
– In lesions due to adaptive immune stimulation
– Accompany prolonged inflammation
• Fibroblasts :
– Present in chronic inflammation & healing wounds
 Suppurative inflammation Septic suppurative inflammation
with no evidence of sepsis (Degenerate neutrophils
(Non-degenerate neutrophils) with intracellular bacteria)
3. Proliferative (hyperplastic/neoplastic) lesions
• Produce lumps and bumps & relatively easy to sample
• Inflammation can accompany many tumours
• Neoplasms damage surrounding tissue, high cell turnover
• Approach for neoplasia involves a logical, stepwise approach using general
principles
• QNS ASKED FOR TUMOURS
1. Is the lesion primarily proliferative and, if so, is it
hyperplastic, benign or malignant?
• Differentiation extremely difficult and not straightforward
• Based on nuclear & cytoplasmic features
• Malignancy: anaplasia, invasion, & metastasis
• Metastases can be detected by imaging & cytology e.g.
aspiration of lymph nodes, organ masses
• Anaplasia: neoplastic cells are poorly differentiated &
highly variable in appearance
• Cytological features for malignancy:
i. Numerous & abnormal mitoses
ii. Cell & nuclear pleomorphism
iii. Cytoplasmic basophilia
iv. Enlarged or bizarre nucleoli
v. Multinucleation (especially with variably sized nuclei)
Nucleoli

Multinucleated

binucleus
Mitosis
2. Are the predominant cells round, spindle, or epithelial?
• Round cells = haematopoietic cells & mast cells
• Spindle cells = from connective tissues, muscle, vessels etc
• Epithelial tumours = square to columnar
3. Can the cell types be subcategorised?
• Glandular: duct or acinus arrangement or intracellular
product
• Intracytoplasmic melanin = melanotic tumour
b) Diagnostic cytology as a component of body fluid analysis
1. Body FLuid Analysis
a) Body cavity effusions
• Abdominal, thoracic, pericardial fluids
• Effusion: pouring out of any fluid into a body cavity or tissue,
i.e. excess fluid
• Assess:
i. Gross characteristics
ii. Protein levels (by refractometer),
iii. Total nucleated cell count
iv. Differential cell count (on a smear)
• Assessed as part of an abdominal cavity disease investigation
Effusions can be divided into:
i) Pure transudates
• Occur in hypoproteinemic states & chronic liver disease
• Have low protein & low cells
• Non-inflammatory and uncommon

ii) Modified transudates

• Non-inflammatory in origin
• Modified by addition of protein or cells, e.g. ascites due to chronic
passive congestion, neoplasia
• Common

iii) Chylous effusions

• Commonly in the thorax eg., from heart worm, heart water
• Due to leakage from lymphatics
• Most chylous effusions are milky white
iv) Exudates (high protein and high total nucleated cell numbers)
1) Non-septic exudate
• Develops from a modified transudate
• Due to direct inflammation caused by irritants that usually produce
few toxic changes in neutrophils, e.g. sterile foreign bodies,
ruptured urinary bladder

2) Septic exudate
• Product of inflammation caused by a wide variety of microbes that
are toxic to neutrophils
• Values for protein & total nucleated cells are high

v) Haemorrhagic effusion (recent or long standing)

• Recent haemorrhage will be characterised by clear supernatant on
centrifugation
• Later supernatant become red-brown or yellow due to degradation
of RBCs
• Erythrophagocytosis & haemosiderin in macrophages seen
b) Synovial fluid analysis
• Conditions: arthritis
• Diagnosis- physical, radiological and laboratory findings
• Synovial fluid is a dialysate [dialysis] of plasma to
which mucus is added by synovial cells

Normal gross characteristics

i. Volume: 0.01 - 1 ml for the dog (<cat, >cattle)
ii. Colour: colourless (light yellow- horse)
iii. Transparency: clear
iv. Viscosity: viscous (hyaluronic acid content)
v. Mucin content: determined by the mucin clot test –
see notes
c) Cerebrospinal fluid evaluation
• CSF: produced by diffusion from plasma and active secretion from choroid
plexus & ependymal linings
• Collected from subarachnoid space & lumbosacral spaces

Laboratory evaluation of CSF

i) Physical Examination
1) Colour: colourless
• Xanthochromia (yellowing of the CSF) in jaundiced animal or previous
haemorrhage in the CSF
• Red CSF = recent haemorrhage (pink to orange supernatant) or
haemorrhage at the time of collection (clear supernatant).
• Grey-green CSF may indicate suppuration

2) Turbidity – clear
• Turbid = excess cells or perhaps protein or bacteria

3) Coagulation
• Clotting may be due to inflammation or haemorrhage
Sampling CSF in monkey
ii) Total nucleated cell count
• Total nucleated cell count (usually done on the EDTA tube
(degeneration of cells within hrs)
• Pleocytosis (an increase in the number of nucleated cells in
CSF):
– Primary inflammatory conditions (trypanosomiasis),
– Differential cell count needed - cytospin
iii) Differential cell counts
• Normal: small lymphocytes (around 60-70%) and
monocytes/macrophages (20-30%)
• Abnormal: neutrophils, neoplastic cells

iv) Protein estimation

• Normal CSF has a low level of protein mostly albumin
• Increases due to inflammatory conditions involve increases
in globulins
d) Respiratory washes
Sampling techniques
• Bronchoaveolar lavage (BAL)
– via a bronchoscope or catheter
– Good for cellular samples of the lower respiratory
tract (alveolar spaces and smaller airways)
• Transtracheal aspiration (TTA)
– provide sterile samples for microbiological
investigation
Types of lining cells and other structures present in respiratory washings
• Ciliated columnar epithelium
– small round to oval nuclei situated at the end of the cell not displaying the
plate of cilia
– Cilia are often lost in the washing process and will be found free
• Mucosecretory (goblet) cells
– Large oval to bulging, elongated cells containing numerous large deep-pink
granules in their cytoplasm.
• Bronchoalveolar cells
– Small, round to square cells, often present in clusters.
– Nuclei are usually round and they have moderate amounts of blue-grey
cytoplasm.
• Alveolar macrophages
– Large with obvious cytoplasmic vacuoles or ingested material (e.g. blue
granules of haemosiderin, black-brown granules of carbon
• Mucus
– Pink to light blue
– Occur in a variety of chronic respiratory conditions (inflammatory or
neoplastic)
• Inflammatory cells
– Egs: neutrophils, eosinophils, mast cells and lymphocytes in low numbers
Commonly diagnosed airway and pulmonary conditions on the basis
of respiratory washes
• Divided into pathological processes: inflammatory, vascular or
neoplastic

1. Acute (active) inflammation

• High levels of neutrophils and lesser numbers of alveolar
macrophages.
• Related to bacterial infection

2. Allergic or parasitic (hypersensitivity) inflammation

– Significant numbers of eosinophils.
– Mast cells and neutrophils will be present in variable numbers

3. Mycotic (fungal) inflammation

– Eosinophils prominent
– Purulent responses may occur. Yeasts may be present in Cryptococcus
neoformans infection
– Fungal hyphae may be present in Aspergillus spp infection.
4. Pulmonary haemorrhage
• Can occur in most conditions affecting the lungs
• Common in: trauma, heart failure, bleeding disorders, neoplasia
• Erythrophagocytosis visible - acute
• Haemosiderin-laden macrophages - chronic haemorrhage

5. Chronic respiratory disease

• Mucin and increased numbers of macrophages common
• Goblet cell and general epithelial hyperplasia

6. Primary and secondary neoplasia

• Neoplasia of the lungs cannot always be diagnosed on respiratory
washes
• Primary epithelial lung tumours more common than metastatic
tumours
• Fine needle aspiration or biopsy of the pulmonary tissue is usually
required to diagnose neoplasia
 Epithelial Cells

Sebaceous cells Squamous cells

(Sebaceous adenoma or hyperplasia) (Squamous cell carcinoma
with suppurative inflammation)
 Mesenchymal Tumors

Spindle Cells Adipocytes

(Hemangiopericytoma) (Lipoma)