FNA and cytoloy sampling techniques

cytology

it’s the examination of cells

• Aspirate smears

• Fluid smears

• Impression smears

• Tape strips

what Cytology is not

• Histopathology is not cytology

• Cytology does not give you the architecture of the tissue

• Cytology may not reveal the precise location of processes

• e.g. It can reveal that there is inflammation in the liver

• - but not if that is portal or lobar (Another example we can see higher number ofinflammatory cells but not where exactly it is happening → so you then take a biopsy as an example)

» Slides CSF and Spinal cord of dog with SRMA (steroid responsive meningitis arteritis)- image - high neutrophil inflammation present only on meninges and blood vessels, not in CSF

why do cytology

» Rapid results- can be done patient side

» Provides information on the nature of the lesion

• May see organisms before culture growth

• Can identify the type of cells present in a neoplasm- which may inform surgical approach

— Extent of margins (do they need to be wide and deep)

— Consider if more than one surgery may provide best outcome (large masses)

— Inform if it may be complicated e.g. mass likely to be attached to other structures, or highly vascular

» Consider what questions you want answering before you take the sample

• Inflammation vs Neoplasia

• Benign vs Malignant

collecting samples for cytology

» Fine needle aspiration (FNA)

• Free needle (no suction)

• With suction

» Fluid aspiration

• ‘Normal’ fluids – e.g. CSF, Joints, Urine

• ‘Abnormal?’ fluids – e.g. peritoneal (small amounts expected in health), thoracic, pericardial

» Flush/Washes

• Tracheal or Bronchoalveolar lavage

• Prostatic wash

» Impression smears

• Directly from exposed surfaces on the skin

• From cut surfaces of organs/biopsies

» Tape strips or skin scrapes

» Swabs

preparation for sampling

» Make sure you have all the equipment to hand

• Needles (21G- green or 23G- blue)

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

» 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

fixative-A fixative is basically a chemical "pause button" for living cells.

When cells are removed from the body, they instantly start to die, rot, and fall apart. If you put them under a microscope like that, they just look like a blurry, mushy mess.

A fixative is a liquid (like alcohol or formaldehyde) that you drop onto the cells to freeze everything exactly as it was when it was alive.

FNA - fine needle aspiration - take it like a woodpecker - quick peck-not supposed to waddle it around

» 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 aspiration

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’

get your fingers around mass to feel it first to help guide you

usually skin mass

smear preparation

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

» Wet fresh preparations smear more readily - otherwise if dry more likely to break the cells

» If in fluid cells may alter in vitro - so usually take fluid out cos cells can swell up cos osmosis, diff impression of what the cells acc look like in patient

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

fluid preparations

» May use the same technique for smearing as solid mass aspirates- just place a drop of well mixed fluid (gently inverted – not shaken) in the same area that you would place the sample and spread in the same method

» Alternative spreading

• Line preparation- blood smear technique but abruptly stop and lift up the spreader smear

• Starfish preparation - if liquid is gloopy so you can take a needle gently raise the top slide and spread out the liquid

fluid preparations

» May use direct smear preparations

» May also use concentrated preparations

• Centrifugation of sample and sediment preparation

• Cytospin preparation

impression smears

» Swabs

• May wish to lightly moisten the swab with sterile water

• Rub over the area to be sampled

• Roll the swab along the length of the smear- may repeat the rolling

» Impression smears from ulcerated external lesion

• Lightly press the slide 2-3 times on the area – moving the slide to a new area each time

• Then clean/surgically prepare the area and either use a scapel to reach the deeper layers, or FNA but via a non-ulcerated surface

» Impression smears from tissue biopsy

• Make a fresh cut surface of the tissue

• Handling the tissue with forceps – lightly blot the cut surface on a tissue to remove excess blood

• Press the tissue lightly to the surface of the glass slide

• Sometimes lightly rolling helps exfoliate cells

ear swabs

tape strips

» Place the tape over the area to be sampled

» Then attach the tape to the slide in a large loop

» Stain the tape by dipping the area that has been applied to the patient in the stain

» Then apply the tape over the slide, with the stained sample area on the surface of the slide, covered by the other side of the tape.

labelling smears

» If you can persuade your practice to buy the smears with frosted ends this makes labelling simpler

» Pre-staining best to use pencil- ink and sticky labels can come off during staining

» When preparing in house/ submitting to the laboratory always best to label

• Animal species

• Animal name

• Unique identifier (Surname or patient no)

• Site sampled

• Date

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!)

» Veterinary Diagnostic Samples will usually fall within UN3373 Category B and require packaging according to code 650

» 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

staining

» In house rapid Romanowsky stains are available

• Modified Wrights/Giemsa/Giemsa-Wright

» Stain maintenance

• Always keep the lids on the stain

• If there is a scum on top then use filter paper to remove it before putting the slide in

• Refresh the stains regularly (approx. monthly if regularly used)

• Consider having separate stains for skin/ear dermatology samples and other sites

• Do not get water in the stains!

» Stain use- manufacturers instructions

• Stain 1 – pale blue- fixative step- can leave slide in here for 1minute or more- useful if thick

• If aspirated fat then may dissolve as fixative usually alcoholic in nature

• Stain 2- manufacturers recommendations

• Stain 3- manufacturers recommendations- although can add a few more seconds if the stain is getting old or sample is very thick.

• Rinse in water- either in a jar- or if under the tap direct the water to the back of the slide and let it run around to the front

adequacy of the smear

» Good cellularity

• Ensures you have good representation of the area sampled

» Poor cellularity

• May be appropriate for some sites

• Frequently will indicate inadequate material for assessment

adequacy of the smear

» Good cellularity

• Ensures you have good representation of the area sampled

» Poor cellularity

• May be appropriate for some sites

• Frequently will indicate inadequate material for assessment

adequacy of the smear

» Adequate tnumbers of cells need to be intact, and well spread out

» Too few intact cells- often due to too much pressure when spreading

• Insufficient material to make a reliable judgment

» Too thick a preparation- too little pressure when spreading

• Unable to really examine the cells to make a reliable judgmen

inflammation vs neoplasia

» May depend on site and presentation

» Inflammatory cells – haematopoietic leukocytes

» Neoplasia

• Epithelial- cuboidal or angular – often tightly adhered

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

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

second pic

» Granulocytes or mixed inflammatory cells most often inflammatory response

» Predominance of a single round cell population- concern for neoplasia

• Mast cell neoplasm – skin- canine

benign or malignant

» Neoplasia is disruption of normal control of the cell cycle

» Malignancy- more aggressive, faster course, more atypia in appearance in most tissues

» NB some tissues may have significant atypia in response to local inflammation

• Fibrocytes

• Mesothelial cells

in pic nucleus is large, loads of nucleoli, also weird shape

cytology

» Examination of individual cells by microscopy

» Rapid, can be patient side, relatively inexpensive (compared to other tests)

» Cannot provide information on tissue architecture, so may not be able to be completely definitive in diagnosis, may not be able to determine the extent of the tissue damage