Approach to the cytology smear

when evaluating a cytology smear

Is there adequate material?

• Some sites may not have an expectation of material, in which case an absence of material may be consistent

• However one should always consider whether the sampling has been adequate- and the absence of material is a true reflection of the site sampled

If cells are present are they well distributed and well preserved?

• If few intact cells then may not be able to assess adequately

• If cells are thickly or densely clumped (vs well spread as single cell layer) then may be more difficult to assess

Is there excessive amounts of debris or ultrasound gel

• Excess amounts of debris may affect staining and interfere with evaluation

• Ultrasound or lubricant gel generally stains brightly eosinophilic, can obscure the smear, and may contribute to poor cell preservation

is there sufficient material on the slide?

» When evaluating a cytology smear

What cells are present?

• Inflammatory cells

• Tissues cells

• Mix of inflammatory and tissue cells

• Are the cells those that are expected for the site sampled?

Are there infectious agents present?

• Could these be ‘bystanders’ or contamination?

• Are they genuinely part of the process sampled?

Are there any other features that may be significant for interpretation of the sample?

preparation for sampling

» Make sure you have all the equipment to hand

• Needles (21G or 23G)

• Glass slides- wiped down with a tissue to remove grease or particles (do not use alcohol at this stage)

» Clean and/or sterile prep the area of entry to the mass/lesion/organ as appropriate

• Some areas such as joints, spinal, or other internal cavities may be particularly at risk for introduction of infection by the sampling procedure- surgically sterile prep these areas

• For sites where we are looking for presence of organisms e.g. swabs of the skin, impression smear of an open lesion – pre-preparation of the area may not be appropriate.

» Scrub hands or use gloves

FNA

» Occasionally also termed FNB – fine needle biopsy

» Free needle

• Only the needle is used- unattached to a syringe

• Best to try initially- if a good quality sample is obtained then no need for suction

• Optimal for ‘delicate’ cells such as lymphocytes (i.e. lymph nodes)

» With suction

• Used when cell yield is low

• Aspiration with a syringe while the needle is in the mass

• May be needed more often for some cell types e.g. spindle cells

Needle collection or Aspiraton

» Firmly hold the mass in one hand (if accessible)

» Use small gauge needles 23G (blue) 21G (green) most often

» Place the needle into the mass and

• Either rapidly move the needle back and forth (‘woodpecker’ action)

• Or pull air into the syringe and then redirect the needle within the mass

— May need to release suction between areas of redirection

• When you see material in the hub of the needle then likely adequate sample

» NB Do not pull the needle out of the mass until you are ready to put the sample on the slide

» ALWAYS release suction before pulling the needle out of the mass

expiration of sample onto the slide

» If there is a syringe attached to the needle remove it first

» Aspiration about 2-3mls of air into a syringe and attach it to the needle with the sample

» Place the needle bevel so that the long side is uppermost and the angled slide is below, and location is about 1/3 in from the edge of the smear.

» Rapidly force the air through the syringe so that the sample is expelled onto the slide in a small area

spreading of the sample

» Place one slide over the other at right angles

» Gently pull the two slides apart

» Minimal pressure on the slides,

• too much pressure -> cell rupture-> non-diagnostic sample

problems with aspiration

» Poorly exfoliative (can reflect the nature of the mass)

» ‘Geographic miss’

smear preparation

» Ideally prepare/spread the smear as soon as possible after the sample is obtained.

» Wet fresh preparations smear more readily.

» If in fluid cells may alter in vitro

» Consider if sending to a laboratory that automated stainers may not stain the entire smear

sending off slides

» If sending slides to an external laboratory they need to be well packaged

» Also tightly sealed- avoid sending with formalin samples- formalin fumes will impact staining

» Staining before send off not required unless you are checking you have good cellularity.

» Samples should be sent according to legislated guidelines and labelled as biologic samples

• Generally packaging requirements less stringent for glass slides on their own (won’t leak!)

Biologic Substances

» Samples should always be sent with a submission form including patient details (signalment) and

appropriate clinical history

• In short – why are you sending the sample and what do you hope to find out?

» Consider if there is the possibility of a zoonosis- label the sample container within the package if so.

» Ensure there is a return address on the package

approach to the cytology smear

» Look at the smear before placing on the microscope

• Areas with cells present will be seen as dark blue staining

• Indicates that you should be able to find and evaluate cells under the microscope

» Start by scanning on a low objective to ‘see’ where the cellular elements of areas of interest may be

• It can be simpler to note where there are high numbers of cells and begin evaluating in those highly cellular areas

• Evaluating single cells can be more challenging to provide a reliable interpretation

» Gradually increase the objective magnification when looking at areas with relevant material.

• Some cells (eg adipocytes) and infectious agents/parasites (eg Angiostrongylus) may be clearly visible on low power, and harder to spot clearly on higher magnifications

identifying cells

» Inflammatory cells

• Neutrophils

• Eosinophils

• Basophils

• Monocytes

• Lymphocytes

— Plasma cells

— Mott cells (‘constipated plasma cells’)

• Mast cells

» Inflammatory cells

• Neutrophils

• Eosinophils

• Basophils

• Monocytes

• Lymphocytes

• Plasma cells

—Mott cells (‘constipated plasma cells’)

— Mast cells

» Neoplasia

» Round cells

• Histiocytoma

• Histiocytic sarcoma

• Lymphoma

• Myeloma

• Mast cell tumour

cell types

» Round cell (often haematopoietic in origin) – round, individual or discrete

» Epithelial- cuboidal or angular – often tightly adhered

• Normal -> Hyperplasia -> Adenoma -> Adenocarcinoma/ Carcinoma

» Spindle cell – spindle or elongated – occasionally stellate, may be adhered, often poorly exfoliative and individual cells

• Normal -> Fibroplasia -> e.g. Fibroma -> Sarcoma

Evaluation of inflammatory cell types

» If inflammatory cells are noted it is important to

Establish if there is significant haemorrhage present

• Could the leukocytes merely reflect blood presence

• Compare ratio of erythrocytes to inflammatory cells: Can also compare to concurrent blood smear from patient if available

Establish the proportions of the cells types present and whether they may be increased for the site

• Differential count

• Observe the number of cells per x50 or x100obj hpf (high power field)

Determine if there are specific morphologic features of the cell types

• Degenerate or non degenerate neutrophils?

haemorrhage

» Haemorrhage may be

• Iatrogenic- occurred at time of sampling eg nicked a capillary when sampling tissue

• Pathologic – part of the process which you are evaluating for your patient

» Iatrogenic haemorrhage will often have platelets admixed

» Prior haemorrhage

• Erythrophagia of macrophages on fresh smears (erythrophagia can occur in vitro with fluids)

• Haem pigment – within macrophages or in the background

toxic and degenerate neutrophils

» Toxic change noted on haematology smears

» Indicates acute demand/accelerated

haematopoesis

» Most often seen in response to inflammation

• eg in response to infection

» Cytoplasmic changes

• Basophilic cytoplasm

• Doehle bodies

• Foamy or vacuolated cytoplasm

• (rare) atypical granulation

» Degenerate change noted on cytology preparations

» Indicates (most often) exposure to endotoxins

» So commonly seen with gram negative bacterial infections

» Nuclear changes

• Moth eaten appearance

neutrophilic inflammation

» Response to infectious agents

• Bacterial

• Fungal

• Parasite

» Response to sterile irritants

• Urine

• Bile

» Response to chemical or trauma damage to tissue

» Response to neoplasia

• May be response to eg ulceration of mass with secondary infection in skin tumours

Lymphocytic inflammation

» Uncommon for lymphocytes to be the primary response

• Generally small lymphocytes would be expected

• May be seen with regression of histiocytoma

• Care with interpretation in the skin, as lymphoproliferative disorders may have a predominance of small lymphocytes- BIOPSY may be required to properly assess

Eosinophilic Inflammation

» Response to parasites

» Response to neoplasia

» Idiopathic eosinophilic disorders

Macrophagic inflammation

» Often admixed with neutrophils

» On cytology likely to reflect pyogranulomatous inflammation- but formation of granulomas is an architectural observation

» So cytologically termed neutrophilic –macrophagic inflammation

» May be seen in response to

• Bacteria

— Consider mycobacterium where appropriate

• Fungi

• Foreign body reactions

Staining

» In house rapid Romanowsky stains are available

• Modified Wrights/Giemsa/Giemsa-Wright

» The in house stains are more aqueous based

• This can affect staining properties of some cells and materials

• Mast cell granules may not be reliably stained by the in house ‘rapid’ stains

• The laboratory automated stainers with a more alcohol based stain may provide better granule staining

summary

» Important to be aware of the site sampled

• Expected cellular elements

• Expected cellularity

» Evaluate if you have adequate material on the smear to make a reliable interpretation

» Determine if you have an inflammatory response, hyperplastic/dysplastic tissue or neoplasia

• Or a combination of the above

» Identify if you have necrosis

» Identify any infectious agents

» Draw a conclusion from your findings- and determine next steps (which may be additional sampling)