Cytology

What are the advantages of cytological sampling?

relatively non-invasive

can often perform on a conscious animal

minimal risk

can sample multiple sites easily

relatively inexpensive

What are the disadvantages of cytological sampling?

small sample obtained - might not be representative

high chance of taking a non-diagnostic sample

What does a fine needle (aspirate) biopsy involve?

obtaining a small sample of cells from inside organs or masses either with or without aspiration

What will you need for a fine needle (aspirate) biopsy?

22 gauge needle, 2 clean slides, 2.5-5 ml syringe, gloves

Method to perform a fine needle biopsy without aspiration

1. put on gloves

2. prepare the skin over the mass aseptically

3. secure the mass with your non-dominant hand

4. insert a 22 gauge needle into the area of interest up to the hub

5. slightly withdraw the needle and redirect it several times within the tissue

6. remove the needle from the patient and apply pressure to the site

7. fill the syringe with air and then attach it to the needle

8. expell air through the needle onto a slide, holding onto the needle at all times

9. dispose of the needle and syringe

10. place another slide perpendicular to the slide containing the specimen and smear along the slide

Method to perform a fine needle biopsy with aspiration

1. put on gloves

2. prepare the skin over the area of interest aseptically

3. attach the syringe to the needle

4. insert the needle into the area of interest and draw back the syringe to 2/3ml

5. maintain this negative pressure and keep the needle in the tissue while redirecting the needle several times

6. release the negative pressure while the needle is still in the patient

7. remove the needle from the area and apply pressure to the site

8. remove the needle from the syringe

9. draw air into the syringe and then reattach and expel the sample onto a clean slide

10. dispose of the needle and syringe

11. place another slide on top of the slide perpendicular and smear along the slide

Where is an impression smear taken from?

the surface of a highly exfoliative tissue/lesion either on a patient or removed

How do you take an impression smear of a removed lesion?

1. gently blot the surface of the sample to prevent blood contamination

2. press the cut surface of the sample onto the face of a clean slide

3. allow the slide to air dry

4. stain using DiffQuik

What are the advantages of an impression smear?

very low risk and no aseptic preparation of the site required

What is a tape strip sample used for?

to collect surface cells, debris and infectious agents, often from skin conditions to examine in-house

What are the advantages of tape strip sampling?

entirely non-invasive

very low risk

no aseptic preparation required

What does swab sampling involve?

using a sterile cotton bud to obtain a surface cytology sample

When is swab sampling often used?

skin cytology

nasal discharge

ocular discharge

vaginal discharge

fistulae

When is tape strip sampling often used?

to investigate skin conditions

What does a urine sediment analysis involve?

it concentrates the solids in a urine sample to enable a wet preparation to be examined under the microscope

What can be identified using urine sediment analysis?

leucocytes, infectious agents, crystals, abnormal cells, red blood cells

How do you prepare urine sediment?

1. invert the urine sample 5-7 times

2. place 1ml urine into 2 Eppendorf centrifuge tubes

3. place the tubes opposite each other into the centrifuge machine, secure the lid and close

4. centrifuge the sample at 1500rpm for 2/10 mins (depends on machine)

5. remove from centrifuge and pour/pipette off most of supernatant

6. mix the sediment by flicking the bottom of the tube while firmly holding the top

7. pour a drop of the sediment onto a clean slide

8. lower cover slip onto specimen using a needle to prevent air bubbles

9. examine sample under a microscope at increasing magnification

10. look for and record blood cells, other cell types, casts, infectious agents and crystals

What are common crystals to observe in urine sediment analysis?

struvite (coffin shape)

calcium oxalate (dihydrate = squares with crosses, monohydrate = ovals)

calcium carbonate

cysteine (hexagonal)

What are some common cells to see in urine sediment analysis?

granulocytes (WBCs)

fat droplets (look similar to RBCs)

keratinocytes (anucleated)

red blood cells

transitional epithelial cells

Why should you examine your cytological sample after taking it?

to assess the quality of the specimen before sending it off

to form interim diagnosis while awaiting pathologist report

to perform in-house diagnosis

How should you examine your cytological specimen?

examine whole slide methodically at a low power first and evaluate its diagnostic quality

identify an area of good cellularity

increase the magnification to examine and describe the cells (think nucleus, cytoplasm, shape)

identify the cells present

identify any artefacts

What are some possible artefacts you might see in a cytological slide?

squashed cells - cell content everywhere

large clumps of cells that cannot be distinguished

cells curled up due to the sample drying out

sample too thick to look at cells in the monolayer

What are the advantages of a fine needle biopsy?

quick and cheap

no sedation required usually for superficial tissues

What are the disadvantages of fine needle biopsy?

risk of haemorrhage

aseptic preparation required

may need ultrasound guidance

Which urine crystal type is shown here?

strubite

Which urine crystal type is shown here?

calcium carbonate

Which urine crystal type is shown here?

calcium oxalate (dihydrate)

Which urine crystal type is shown here?

calcium oxalate (monohydrate)

Which urine crystal type is shown here?

cysteine

When would you perform a fine needle biopsy without aspiration?

on tissues where cells exfoliate easily or highly vascular tissues

When would you perform a fine needle biopsy with aspiration?

on highly structured tissues which don’t exfoliate well

What is an exampe of tissues you would perform a fine needle biopsy with aspiration on?

muscle and spindle cell masses

What is an exampe of tissues you would perform a fine needle biopsy without aspiration on?

lymph nodes, liver, spleen

Why would you not use a 21 gauge needle for a fine needle biopsy?

they are wider and so more likely to cause haemorrhage

Why would you not use a 23 gauge needle to perform a fine needle biopsy?

they are narrower so likely to yield fewer cells

How do you prepare a slide from a fine needle biopsy for examination?

1. allow slide to air dry

2. stain using standard DiffQuik staining method

How do you take a tape strip sample?

1. measure out a strip of tape a bit longer than your slide

2. attach the tape to the non-frosted end of the slide without touching the underside of the tape (or can just hold it loose)

3. press the centre of the tape several times to the area of interest

4. attach the tape to your slide now if you have not already

5. stain the tape in solutions B and C of the DiffQuik

6. rinse the tape with distilled water

7. place the slide onto some tissue with the sticky side of the tape facing up

8. stick the tape along the slide and blot gently with a tissue

9. leave to dry before examining

Why do you not use solution A when staining tape strip samples?

it will melt the glue on the tape

How do you obtain a swab sample?

1. roll or rub the swab in the area of interest, ensuring all sides of the swab are covered

2. place the swab side down on one end of a clean slide and roll it along the slide to deposit the collected cells

3. if using a sample from a greasy area, heat-fix the slide by warming the underside

4. stain the slide with DiffQuik

How do transitional epithelial cells appear under a microscope?

granular with irregular borders

How do squamous epithelial cells appear under a microscope?

keratinised appear large and angular

non-keratinised appear large with a central nucleus, may be round or angular

How do leucocytes appear under a microscope?

round, colorless and slightly granular, slightly larger than RBCs

How do red blood cells appear under a microscope?

round, smooth and biconcave

What is the most common crystal to see in urine?

struvite

Which species is it common to see calcium carbonate crystals in the urine of?

horses, rabbits and goats