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Inflammation
Hyperplasia/dysplasia The following are the general categories of cytologic
Neoplasia interpretation:

Non-diagnostic
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Inflammation
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Hyperplasia/dysplasia
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eClinPath Home Note: Often more than one category is present, as
Diagnostic challenge inflammation can result in dysplastic changes in the
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surrounding tissue and inflammation often accompanies a
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neoplastic process.
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Hematology
Hemostasis Non-diagnostic samples
Urinalysis
Chemistry There are many reasons for obtaining a non-diagnostic sample:
Cytology
Overview Poor cellularity of the sample: Due to a poorly exfoliating
Sample collection lesion or poor sample collection.
Cytologic patterns Excessive blood contamination: This contributes
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leukocytes, which need to be differentiated from a true
Effusions
inflammatory infiltrate, which can be difficult. When we
Synovial fluid
Bone Marrow describe cellularity, we usually do not include blood-
 Educational videos associated leukocytes. They do not help with
Exotics interpretation and can hinder it.
Many smudged or ruptured cells: This may result from
Advanced Search exuberant collection methods (e.g. excessive pressure
put on the syringe during aspiration) or smear
preparation (excessive pressure put on the spreader slide
– squashing cells), though some tumor cells are
excessively fragile and prone to rupture.
Sampling error: Aspiration of surrounding fat or another
structure, e.g. aspiration of the salivary gland when
attempting mandibular lymph node aspiration.

If the sample has adequate cellularity and the cells are well-
stained and well-preserved, the next step in cytologic diagnosis
is the identification of the cell types and pathologic process
that are present. It helps to ask a series of questions when
working through smears:

Is there evidence of hemorrhage?

Does the smear contain inflammatory cells in excess of
blood?

Can a cause for the inflammation be identified?

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Are there tissue cells present?

Are they expected normal for the aspirated site?

If the cells are not expected for the aspirated site, what is
their lineage – epithelial, mesenchymal, round or discrete
cell, endocrine/neuroendocrine?

Are they neoplastic or non-neoplastic (e.g. cyst)?

Do the cells show cytologic criteria of
malignancy?
What other features are present, e.g. erythrophagia,
necrosis, mineralization.

No cytologic abnormalities
Cells are present in normal numbers for the tissue aspirated
and do not possess significant criteria of malignancy. This
finding is most common when aspirating internal organs or
lymph nodes, as most skin and subcutaneous masses
represent a true pathologic process.

Inflammation
Inflammatory responses are classified by the type of
inflammatory cells within the lesion, which also gives us clues
as to the cause of the inflammation. Note that the causes listed
below are not exhaustive lists, but more common examples.
 Neutrophilic inflammation Degenerate neutrophils

Suppurative
>85% neutrophils
The appearance of neutrophils may be helpful in
identifying cause.

Non-degenerate neutrophils (resemble those in

blood with condensed clumped chromatin):
Causes include immune-mediated conditions,
sterile irritants (bile, urine), bacterial infection,
protozoal or fungal infection. The lack of
degenerative change in neutrophils does not mean
that the inflammation is not due to bacterial
infection. Some species of bacteria will not cause
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this morphologic change in neutrophils.

Degenerate neutrophils: These are undergoing
karyolysis and taking up water, resulting in cell and
nuclear swelling. In these cases, suspect bacterial
sepsis and look carefully for phagocytized
organisms. However, such swelling can also be an
artifact of sample collection into fluids and
storage. Degenerate neutrophils do not always
mean bacterial sepsis and can be seen with fungal
infections and chemical irritants.
It is always a good idea to culture a sample with
neutrophilic inflammation, unless you know the cause is
not bacterial sepsis, e.g. pancreatitis.

Granulomatous inflammation Hemosiderophages

Histiocytic/macrophagic
 Macrophages dominate in these lesions and they may
take on various appearances, depending on the cause,
e.g. epithelioid, multinucleated, vacuolated and
phagocytic (“reactive”). Epithelioid macrophages are
generally not phagocytic or vacuolated and mimic
epithelial cells. The presence of multinucleated
macrophages supports a granulomatous inflammatory
response.

Causes: Foreign body reactions, fungal infection,

certain bacterial infections, such as
Mycobacterium.
Macrophages in cytologic smears may show cellular
phagocytic activity.

Erythrophages: This indicates recent hemorrhage

(<24 hours). Note that erythrophagia can also
occur as an artifact during storage of fluid
specimens (e.g. body cavity fluids). Macrophages
retain phagocytic activity in vitro.
Hemosiderophages: These are macrophages that
contain hemosiderin in their cytoplasm. The
hemosiderin is from the iron component of the
porphyrin ring of hemoglobin (from phagocytized
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erythrocytes) and represents the storage form of

iron. Hemosiderophages supports prior
hemorrhage (>24 hours duration as it takes time
for hemosiderophages to form).
Melanophages: These macrophages contain
phagocytized melanin in their cytoplasm. They can
be a normal finding in lymph nodes but can also
represent drainage from a melanoma in these
organs.

Mixed
This is comprised of a
mixture of neutrophils and
macrophages, with
neutrophils being typically
non-degenerate, unless
there is concurrent
bacterial sepsis.
Mixed inflammation
Neutrophils usually
dominate, but may not if
the outer edges of the lesion are aspirated (the latter
areas may be more macrophage-rich). This type of mixed
inflammation is also called pyogranulomatous,
particularly if there are multinucleated macrophages.
 Lymphocytes and plasma cells can be seen in low
numbers well.

Causes: Foreign body reactions (e.g. keratin from

a furunculosis), fungal infection, longer-standing
bacterial infection, infection with specific bacteria
(e.g. Nocardia, Actinomyces). Chronic tissue injury
can also incite a mixed inflammatory response.

Eosinophilic
This is generally defined as
inflammation consisting of
more than 10-20%
eosinophils, although cut-
offs vary between clinical
pathologists, with some
pathologists indicating an
Eosinophilic inflammation
eosinophilic component to
the inflammation if <20%.

Causes: Hypersensitivity reactions to allergens or

infectious agents such as parasites, certain fungi
and foreign bodies (e.g. keratin), insect bites,
cancer (e.g. mast cell tumors).
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Lymphocytic or lymphoplasmacytic
This consists of a mixture of mostly small lymphocytes
along with plasma cells. Other inflammatory cells, such
as “activated” macrophages may be seen as well.

Causes: Antigenic/immune stimulation, e.g. tick

bite, viral infections or chronic inflammation.

Hyperplasia/dysplasia
The strict definition of hyperplasia
is an increase in the number of
cells in a tissue; however, the term
is often used in a more generic
fashion in cytology as a non-
neoplastic enlargement of a
tissue. Hyperplasia is often the
result of hormonal influences (e.g.
Dysplasia
benign prostatic hyperplasia,
perianal gland hyperplasia), tissue
injury (e.g. regenerative nodules in the liver, granulation tissue
with fibroplasia) or antigenic stimulation (lymphoid
hyperplasia). Aspiration of hyperplastic lesions may result in a
higher than expected cellularity and cells may display some
 mild criteria of malignancy, such as a mildly increased N:C ratio,
darker blue cytoplasm, slightly more prominent nucleoli or finer
chromatin than normal. The latter change can be referred to as
“atypia” or “dysplasia”.

Dysplasia, or disordered growth, is most often seen in epithelial

tissue secondary to inflammation or irritation. Dysplasia results
in loss of uniformity of the individual cells and disordered
architectural arrangement of the cells. It also results in atypical
cytologic features in the aspirates cells, including nuclear to
cytoplasmic asynchrony, increased cytoplasmic basophilia,
anisokaryosis and anisocytosis. Dysplasia can be cytologically
difficult to distinguish from neoplasia as dysplastic lesions
often contain more criteria of malignancy than strictly
hyperplastic lesions.

Although hyperplasia and dysplasia are non-neoplastic

processes, they likely represent a continuum with benign
neoplasia. Cytologically, a hyperplastic process can be difficult
to distinguish from a benign neoplastic process and if there is
significant dysplasia within the tissue, one must exercise
caution as to not interpret dysplasia as malignant neoplasia.
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Histopathologic assessment of the tissue should always be

done if there is any doubt.

Neoplasia
Neoplasia is suspected when there is a population of tissue
cells in an aspirate that are not expected to be there, there is a
mass lesion consisting of cells that are in excess for the
aspirated site (e.g. too many sebaceous cells in a skin lesion)
or the tissue cells are showing cytologic criteria of malignancy.
Neoplasms are further divided into four general tumor
categories: Epithelial, mesenchymal, discrete (round) cell and
endocrine/neuroendocrine (naked nuclei) neoplasms, based on
their cytologic features and pattern of arrangement.

Epithelial neoplasms
Epithelial tumors are cohesive and
form clusters or sheets. They can
show trabecular, circular to
papilliform arrangements. Acini
may be seen in cells that produce
secretory product. Examples of
epithelial tumors include perianal
gland adenoma, transitional cell
Epithelial cells
carcinoma, biliary carcinoma,
 squamous cell carcinoma. Epithelial cells generally have the
following features:

Large, round to polygonal cells

Distinct cell borders
Tightly adherent to each other
Round to oval nuclei that can be basilar in columnar cells
or eccentric in other cell shapes

Epithelial tumors can be benign (adenoma) or malignant

(carcinoma). Benign versions consist of well-differentiated cells
that can be difficult to distinguish from normal tissue (unless in
excess for the aspirated site) or hyperplastic lesions (which
may require evaluation of tissue architecture, e.g. normal
architecture and arrangement around ducts would indicate
hyperplasia versus neoplasia for skin adnexal tumors).
Malignant epithelial cells usually demonstrate cytologic criteria
of malignancy particularly as they become more aggressive or
advanced. However, some carcinomas (e.g. the rare perianal
carcinomas) do not always show features of malignancy but
behave in a malignant fashion. As carcinomas become less
differentiated they lose some of these features and often
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become less cohesive, mimicking mesenchymal cells. Indeed,

epithelial tumors undergo epithelial-to-mesenchymal transition
when they are metastasizing so spindled cells within an
epithelial tumor may represent the tumor undergoing EMT or
reactive fibroplasia (e.g. schirrous response, which is seen with
certain epithelial tumors such as intestinal tumors). In the more
anaplastic tumors, immunohistochemical staining for
cytokeratin can be necessary to confirm an epithelial origin. We
can often determine the type of epithelial tumor on cytology,
particularly if the cell of origin has specific features (e.g.
perianal tumors have a “hepatoid” appearance; exocrine
pancreatic carcinomas have faint pink cytoplasmic granules) or
the tumor cells are showing features of differentiation (e.g.
squamous cell carcinoma). In many cases, the type of tumor
may be based on the aspirated site, e.g. mammary gland, along
with cytologic features (i.e. we use the history to help determine
what the lesion or tumor is).

Mesenchymal neoplasms
Mesenchymal neoplasms carry features of their embryonic
tissue of origin, the mesenchyme. The cells are generally
individualized and spindled in shape. They can be seen in
aggregates (not clusters), often held together by extracellular
matrix. They do not typically demonstrate cell-to-cell adhesion.
Due to increased matrix production, there are some
 mesenchymal tumors (e.g.
fibroma) that do not exfoliate well
and aspirates may be of low
cellularity making a definitive
cytologic diagnosis difficult.
Examples include myxoma,
fibrosarcoma, osteosarcoma,
melanoma and
Mesenchymal cells
hemangiosarcoma. Mesenchymal
tumors generally have the
following features:

Spindle, oval or stellate-shaped cells

Indistinct cell borders, that taper into the background
Round to oval to elongate nuclei that are usually centrally
located
Cells are scattered individually or in aggregates, usually
within matrix.
Less cellular than the other tumors due to matrix
Matrix can be present in the background as well as
within aggregates
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As for epithelial tumors, mesenchymal tumors can be benign

(“..oma”) or malignant (“sarcoma”). Some types of
mesenchymal tumors, e.g. soft tissue sarcomas, are called
sarcomas, even though they do not metastasize quickly. They
are, however, locally invasive. There are also certain types of
mesenchymal tumors that mimic epithelial tumors, showing
cell-to-cell adhesion, including melanoma and epithelioid
variants of hemangiosarcoma. It can be quite difficult to
differentiate between a mesenchymal tumor and reactive
fibroblasts on cytologic features alone, because the latter can
have quite large nuclei and prominent nucleoli (they also display
moderate to occasionally marked anisokaryosis and
anisocytosis). This is particularly a problem in bloody or poorly
cellular specimens. Several variants of mesenchymal tumors
can be diagnosed with certainty on cytology if they have
characteristic cellular features (e.g. crown-like cells in soft
tissue sarcoma, melanin in melanocytic tumors) or
characteristic matrix (e.g. osteosarcoma, chondrosarcoma). We
can also use site to narrow down the differential diagnostic list.
For example, a gastrointestinal stromal cell tumor or smooth
muscle tumor (leiomyoma, leiomyosarcoma) would be
suspected for a mesenchymal tumor arising in the intestine.
However, there are many mesenchymal tumors in which a
definitive determination of tumor type cannot be provided, even
when we know the aspirated site.
 Discrete (round) cell neoplasms
Discrete or round cell tumors often are of hematopoietic origin
(lymphoma, histiocytic, mast cell tumor) and as the term
suggests, consists of individualized round cells. Cells tend to
exfoliate readily and aspirates are often of high cellularity. We
can use morphologic features of the cells, including the
presence or absence of granules and cytoplasmic and nuclear
features, to determine the type of round cell tumor.

Mast cell tumor

These are readily

recognized by the presence
of purple cytoplasmic
granules.
They also have round
eccentric nuclei with
smooth chromatin. The Mast cell tumor

nuclei can be hard to see as

the granules soak up the stain

The degree of granularity varies between tumors.

Granules may be harder to discern with water-
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based stains, such as Rapid stains, particularly in

the less well-granulated tumors.
Low grade tumors are typically well-granulated.
Higher grade tumors can be poorly or well-
granulated and nuclear criteria of malignancy
(nuclear atypia, binucleation, large nuclei, mitotic
figures) are more reliable than granularity for
determining the grade of mast cell tumors on
cytology. Tumor grading for dermal (not
subcutaneous) mast cell tumors in dogs is best
done by histopathology.

Histiocytoma: Cell of origin is the

epidermal Langerhans cell. These
may not be tumors per se as they
regress without treatment due to a
cytotoxic T cell immune response.
They can be single (usually are) or
multiple

Round to oval with variably Histiocytoma

distinct cytoplasmic
borders.
Moderate to abundant amounts of clear to light blue
cytoplasm
 Nuclei are eccentric and round to oval to indented
Nuclei have finely stippled chromatin and nucleoli are not
apparent
Cells are often found dispersed within a moderately blue
background
Minimal cellular atypia, uniform cell size and morphology
– they have a bland appearance
Regressing tumors are associated with increased
numbers of small lymphocytes (tumor infiltrating
cytotoxic T-cells)
Note: Histiocytomas generally consist of very bland,
minimally atypical cells. If a high degree of cellular
atypia (numerous criteria of malignancy) are found and a
histiocytic lineage is still suspected, histiocytic sarcoma
should be considered a differential diagnosis.
The main differential diagnosis is an extramedullary
plasmacytoma. Lightly stippled chromatin, abundant
light blue cytoplasm, indented nuclei and the blue
background are used to distinguish between these
lesions (not all features may be present in every tumor).

Plasmacytoma: These arise from

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plasma cells, which form tumors

(usually solitary) in extramedullary
sites, such as the skin (digit, ears,
mouth) in dogs.

Round to slightly oval cells

Distinct cell borders
Variable amounts of blue Plasmacytoma

cytoplasm (often deep

blue), some have perinuclear clear zones
Nuclei are round, occasionally oval, and eccentric
Nuclei have clumped chromatin and nucleoli are not
apparent
More atypia (anisocytosis and anisokaryosis) than
histiocytic tumors
Binucleation and, occasional, multinucleation is
common. Multinucleated cells may show marked
intracellular anisokaryosis
Amyloid may be present in skin tumors.
The main differential diagnoses are a histiocytoma or
plasmacytoid variants of lymphoma. Compared to a
histiocytoma, the cells have more distinct boundaries,
darker cytoplasm, rounder nuclei (even in multinucleated
cells) and clumpier chromatin. They may have
perinuclear clear zones. With plasmacytoid variants of
 lymphoma, cells with higher nuclear to cytoplasmic
ratios resembling lymphocytes are expected to be
present.

Lymphoma: There are multiple

forms of cutaneous lymphoma,
including inflammatory,
epitheliotropic and non-
epitheliotropic forms. In the skin,
lymphoma can consist of solitary
to multiple nodules, plaques or
ulcerative/exfoliative lesions. It
Cutaneous lymphoma
can be pruritic. Lymphoma, as we
know, also arises in other sites.
They are usually of T or B cell origin. Lymphomas of natural
killer cell origin are quite rare. Lymphoma is most easily
recognized when it consists of large cells or cells that are not
expected in inflammatory lesions, such as many granular
lymphocytes. It is far harder to recognize when the cells are
intermediate to small, however the lack of other immune cells
(plasma cells) or inflammatory cells can lead to a suspected
diagnosis of lymphoma. Lymphoid cells have the highest
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nuclear to cytoplasmic ratios of all the round cells. They can

rupture easily and one can see cytoplasmic fragments
(“lymphoglandular” bodies) in the background, which can be a
helpful but not definitive finding. The main differential
diagnoses for cutaneous lymphoma are inflammatory or
immune-mediated conditions or localized antigenic stimulation,
e.g. pseudolymphoma secondary to an insect bite. There are
histiocytic variants, which have more abundant cytoplasm and
can mimic a histiocytoma. Ultimately, the diagnosis of
cutaneous lymphoma often requires biopsy with
immunohistochemical staining to confirm a T (CD3) or B (e.g.
Pax-5 or CD20) origin for the tumor cells.

Transmissible venereal tumor: This

sexually transmitted tumor is
thought to be of histiocytic origin.
It is mostly seen in warmer cell
climates and is often around the
mouth or genital region, but can
be seen in other sites.

Monomorphic population of Transmissable venereal tumor

round cells
Medium to large round nucleus that is eccentric or
central
 Nuclear chromatin is clumped and mitotic figures are
common
Can see binucleation or multinucleation as well as
nucleoli
The cytoplasm is characteristic: Abundant light blue to
gray with moderate to many discrete margined vacuoles
Can have infiltrates of small lymphocytes

Endocrine/neuroendocrine tumors
These tumors have a
characteristic appearance,
forming packets of cells. Cells
often exfoliate in large numbers
but are fragile and aspirates
contain many bare nuclei from
ruptured cells, hence some people
call them “naked nuclei”
Endocrine cells
neoplasms. They are of secretory
epithelial (producing hormones,
e.g. thyroid tumors) or neuroectodermal origin, with the latter
secreting neurotransmitters, such as epinephrine in
phaechromocytomas. Many of these tumors have quite uniform
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or bland cytologic features, but show aggressive malignant

behavior (e.g. thyroid carcinomas in dogs), therefore cytologic
criteria of malignancy are unreliable and we go by the known
biologic behavior of the tumors. The type of endocrine or
neuroendocrine tumor is generally determined by site, e.g. a
cervical neck mass could be thyroid or parathyroid in origin,
with the former being more common. In some types of tumors,
we can be more definitive, for example thyroid follicular tumors
can contain tyrosine granules (blue green pigment) in the
cytoplasm.

Round to polygonal cells found in cohesive packets or

small sheets
Nuclei are round to oval and central to eccentric
Nuclear chromatin is fine to smooth
Indistinct cell borders

Epithelial Mesenchymal Discrete

E
“round” cell

Typical High Low to moderate High

cellularity of (May be high in a n
 aspirates few specific f
tumors, e.g. soft
tissue sarcoma)

Cell Clusters Individual cells, Individual

Associations (adherent); or in some non- cells; may
can see cohesive form
acinar aggregates aggregate-like
formation if arrangements
secretory in thicker
aspirates

Cell Shapes Variable: Spindled to Round (ish)

Often stellate to rarely
polygonal, round (histiocytic
columnar, or sarcoma)
cuboidal

Cell Size Medium to Medium Small to

large medium; large
with large cell
lymphoma

Cell Borders Distinct in Often indistinct Distinct

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most tumors

Nuclear Shape Round to oval Oval to elongated Round,

to round (e.g. sometimes
soft tissue indented
sarcoma) (histiocytic
tumors)

Other Can see Some tumor Cytoplasmic

Characteristics features of types may be purple
differentiation associated with granules in
(e.g hepatoid characteristic mast cell
cells, extracellular tumors may
squamous matrix (e.g. occasionally
cells) chondrosarcoma) stain poorly
with Diff-Quik

Examples of Benign: Benign:“-omas”, Lymphoma

tumors Adenomas e.g. fibromas Plasmacytoma
Malignant: Malignant or Mast cell
Carcinomas invasive: tumor
Sarcomas Histiocytoma
Transmissable
veneral tumor