Cytology

Describe TWO rules for blood sample collection

1. Fast animals for 6 - 12hr prior to prevent lipaemia

   • → Artificial elevation of Hb, TP, AST and ALT

2. Avoid haemolysis of sample

   • → Interferes with TP, lipase, bilirubin, urea, AST, ALT, ALP, phosphorus and potassium

Fill out the following table

Colour top	Anticoagulant	Mechanism	Use
Red
Purple
Grey
Blue
Green

Colour top	Anticoagulant	Mechanism	Use
Red	None (plain tube)	Clotted blood is spun down to obtain SERUM	Biochemistry (NOT glucose) Endocrine test
Purple	EDTA	EDTA chelates calcium (binds Ca2+ necessary for secondary haemostasis)	Haematology #1
Grey	Potassium oxalate/sodium fluoride	Oxalate = Anticoagulant Fluoride = Prevents glucose utilisation	Glucose assessment
Blue	Sodium citrate	Sodium citrate chelates calcium	Coagulation panel #1
Green	Lithium heparin	Inhibits thrombin formation	Plasma can be used for most biochemical tests Good for urgent biochem as it does not need to clot first Haematology (but interferes with WBC staining) so not best for CBC Bird blood #1 (CBC and biochemistry from SAME small sample)

Serum vs. plasma

Serum = Liquid part of blood that remains AFTER the blood has clotted (contains NO fibrinogen)

Plasma = Liquid part of blood that has NOT clotted (contains fibrinogen and other clotting factors) using an anticoagulant (eg. EDTA)

Fill out the following table

Colour	Cause
Clear
Yellow
Milky white
Red

Colour	Case
Clear	NORMAL cat and dog
Yellow	NORMAL horse, cow, sheep Hyperbilirubinaemia in cat and dog
Milky white	Lipaemia
Red	Haemolysis

Blood Smear Preparation

• When to prepare

• Method

• 6 Common errors

When: ASAP after blood collection (cells break down after removal from the body)

• Ideally use fresh blood with no anticoagulant

• EDTA most common, but may cause artefactual changes

Method:

• Spreader slide = Polished/ground edge slide to allow distribution of blood cells

Errors:

1. Drop size too big or small

2. Not allowing drop of blood to spread along edge of spread slide before smearing

3. Not enough or too much pressure on spread slide

4. Spread too fast/slow

5. Removing spread slide too early

6. Ensure adequate angle of spread slide (too steep = short smear, too flat = long smear)

Cytology

• Definition

• 7 Advantages

• 5 Disadvantages

• 4 Reasons to perform cytology in-clinic

Definition: Harvesting cells or fluid for microscopic examination of INDIVIDUAL cells

+ve:

1. Quick

2. Cheap

3. Samples collected without GA

4. Relatively non-invasive

5. Assessment of tissue AND fluid (cannot assess fluids with histology)

6. Some interpretation in-clinic

7. Superior for round cells

-ve:

1. No appreciation of lesion architecture or its margins (cannot stage a tumour)

2. Small sample that is not always representative (multiple samples required)

3. Difficult to distinguish between neoplasia or hyperplasia (cannot rule out malignancy 100%)

4. NOT ideal for mesenchymal lesions (difficult to distinguish fibroblasts in granulation tissue from neoplastic mesenchymal cells due to lack of architecture)

5. Results are as good as the person performing it (refer samples when in doubt)

In-Clinic:

1. Clinician must have basic skills to ensure specimen is adequate to submit to the lab

2. Allows for faster examination = faster prognosis = faster treatment (advantageous for patient)

3. Local extension of the clinical exam

4. Clinic revenue

Describe THREE methods of cytology collection

• Equipment

• Method

• Indications

ONE: FNA

Equipment: 5 - 10mL syringe, 23 - 25G needle and slides

Methods:

FNI

Needle ONLY (no syringe) is placed into the mass and redirected serval times to obtain cells in the needle/hub

Indication: Delicate cells (eg. lymphocytes) or bloody tissue (eg. spleen)

FNA

Negative pressure technique which aims to dislodge cells and draw into the needle hub

• Needle attached to syringe is inserted into the mass and negative pressure is created while redirecting several times

Indication: Firm or fibrous mass

Indications:

1. 5cm SC mass

2. Fluid in abdomen

3. Enlarged liver

TWO: IMPRESSION SMEAR

Equipment: Slides ONLY

Method:

1. Cut mass and gently dab the cut surface or swab lesion dry to remove blood and exudate

2. Gently dab cut surface ono slide

Indication: Biopsies of solid mass or lesions at necropsy (eg. intestinal mass)

THREE: SCRAPING

Equipment: Scalpel and slides

Method:

1. Cut fresh surface of mass

2. Blot sample dry with paper towel or clean ulcerated surface with saline and dry to remove exudate

3. Scrape surface gently with scalpel at a 45˚ angle OR until blood oozes from the ulcer if on live animal

4. Slide edge of scalpel blade at 45˚ along the glass slide making a trunk

5. Use tip of scalpel blade to make stems ("trunk and stem pattern") which contain a single layer of cells for examination

Indications:

1. Mesenchymal tumour excisional biopsies

2. Shallow ulcerated lesions (difficult to insert needle for FNA) eg. SCC in cats

3. Lesions at necropsy

3 ways to smear a thin fluid sample

1. Smear like blood smear

2. Concentrate sample before smearing by centrifuging to remove supernatant (fluid)

3. Make fluid stop prep (suddenly stop blood smear to create concentrated line of cells)

How to smear a semi-fluid or solid sample

Squash prep: Take additional slide and squash material at a right angle and slide spreader off the end of the slide

5 Tips for preparing slides

1. ALWAYS prepare multiple samples as not all are likely representative of the lesion

2. Label frosted edge with pencil as stains will dissolve pen or marker

3. Only stain 1 or 2 smears for examination before sending rest (unstained) to lab

4. Store adequately

   • Do NOT store slides in fridge (water condensation destroys cells)

   • Dried smears do NOT require immediate staining

   • Place smears in plastic slide holders to send to lab

   • Keep slides away from formalin (separate packaging) as it destroys cells in cytological samples

5. ALWAYS provide history and be precise with the description of the lesion location (eg. thoracic lesion vs. SC thoracic lesion)

Describe the 5 steps of the diff quik staining technique

1. Ensure slide is air-dried

2. Fixative (methanol) = 6 dips and dab off excess (fixes cells to slide)

3. Pink = 6 dips and tap off excess onto paper towel to prevent dilution of purple and make it last longer

4. Purple/blue = 12 dips

5. Rinse off excess stain with water after and allow to dry before examining (can use hair drier to accelerate drying process)

5 Recommendations for diff quik staining

1. Ensure you dip as movement of dye over the cells encourages uptake

2. Can go back and re-dip into pink or purple stain if not enough on initial examination

3. Variable staining intensity is expected due to difference in cell thickness in different areas

4. ALWAYS replace lids to prevent evaporation of stains due to alcohol content

5. Separate stains for ear smears to prevent yeast and bacterial growth in stains

Position of condenser for:

1. Stained slide

2. Unstained slide (eg. urine sediment)

1. Stained slide: Condenser directly under stage

2. Unstained slide: Condenser as far away from stage as possible

Label the following slide artefacts

Squames = Keratinised epithelial cells from the skin of patient or fingerprints of operator Angular and dark blue structures with sharp ends May also be diagnostic (SCC or epidermal inclusion cysts)	Glove Powder (L) = Round/oval structures with which refractile and crystalline with a clear outer edge and a small dark dog in the centre Misinterpreted as Cryptococcus neoformans R = Cryptococcus = Larger purple centre that is more variable in size and NOT refractile	Salivary Gland Epithelial Cells = Collection of salivary glands when aspirating submandibular lymph nodes Pale blue-grey amorphous material in background = mucus
Stain Precipitate (L) = Highly variable sized/shaped chunks of dark purple precipitate which is coarsely granulated Stain is old and has not been filtered or replaced Misinterpreted as bacteria Bacteria (M and R) = Uniform in shape	U/S Gel = Magenta and granular mass that obscured cells	Muscle Fibres = Collection of muscle fibres when aspirating SC Red circle = Striations which are observed as stripes Also collection of liver when aspirating the lungs

What is this?

• Appearance

• Misinterpreted as?

Nuclear Debris

Appearance: Purple strands of nuclear material due to unavoidable damage of cells during preparation

Misinterpretation: Fungal hyphae

How to determine contamination vs. bacterial infection

Assess for inflammatory response to confirm infection

• Organisms within cytoplasm of neutrophils or macrophages are unlikely to be contaminants

CSI-TeaM approach for examination of cytology samples

1. Are there useful cells present?

2. Is the staining adequate?

3. Inflammatory vs. non-inflammatory (neoplastic)?

4. What is the cell type present?

5. Is it malignant or benign?

ONE: ARE THERE USEFUL CELLS PRESENT?

• Method

• Ideal

• 4 Indications to resample

Method: Assess in 4x objective

• Look a the feathered edges where cells tend to aggregate

• Take note of cellular areas of interest to examine in higher power

Ideal: Areas of intact NON-HAEMATOGENOUS cells which are well-spread out into a single layer (should see cell definition)

Resample:

1. ONLY blood

2. No cells or too few to examine

3. Too thick (many cells, but not smeared adequately)

4. Ruptured cells during smearing ALL PRODUCE NON-DIAGNOSTIC RESULTS

TWO: IS THE STAINING ADEQUATE?

• Method

• Ideal

Method: Assess on low or medium power

Ideal:

1. Varying shades of pinks, purples and blues (not just all pink or all purple)

2. Clear differentiation between cytoplasm vs. nucleus vs. background

THREE: INFLAMMATORY OR NON-INFLAMMATORY?

• Method

• 3 Steps after confirming inflammation

• 4 Types of inflammation

• 4 Causes of non-inflammatory sample

Method: High power 40x to assess cellular detail

Inflammation:

1. Describe (type of cells)

2. Identify organisms (± septic)

3. Treatment Do NOT continue with steps 4 and 5 (reserved for non-inflammatory lesions)

Types of Inflammation:

1. Neutrophilic >85% = Suppurative (purulent)

   1. Septic = Suppurative inflammation with bacteria present (within degenerate neutrophils)

2. Mononuclear

   1. Histiocytic = Macrophages (chronic inflammation)

   2. Granulomatous = Epitheliod macrophages and multinucleated giant cells (mycobacteriosis, fungi, parasites, foreign body reaction)

   3. Lymphocytic/plasmacytic (allergy, immune reaction, viral infection, chronic inflammation)

3. ± Eosinophilic >10% (hypersensitivity, parasite migration)

   

Non-Inflammatory:

1. Neoplasia

2. Hyperplasia

3. Cysts

4. Calcium deposits

What are these? What do they mean?

Naked Nuclei = Nucleus with no cytoplasm and appears larger than normal (as do their nucleoli) which may be interpreted as malignancy and should NEVER be interpreted

Does the presence of inflammatory cells exclude the possibility of underlying neoplastic lesion? Why?

No

1. Many neoplasms have inflammation due to secondary infection, response to necrosis within the neoplasm or paraneoplastic response

2. Blood contaminated cells will also have neutrophils NORMALLY

Degenerate neutrophils

Abnormal looking neutrophils with degraded nuclei ± pyknosis (small), karyorrhexis (fragmentation) or karyolysis (fading) which is most common in septic environments

How do distinguish between neoplasia and hyperplasia

Difficult as BOTH usually contain a monomorphic population of cells, therefore ask for a 2nd opinion in the lab

FOUR: WHAT CELL TYPE IS PRESENT?

• 3 Cell types

• How to differentiate

Types:

1. Epithelial

2. Mesenchymal

3. Round

Differentiate:

1. Size

2. Shape

3. Cell borders

4. Clustering/clumping

5. Cellularity of FNA

Name the Cell Type

• Size

• Clustering/clumping?

• Cellularity of FNA

• Morphological appearance

• Cell borders

Epithelial Cells

Size: Large cells

Clustering: Usually cohesive and STICK together in sheets or clusters (can be single too)

Cellularity: Exfoliate easily so FNA or impression smears yield large numbers of cells for an epithelial lesion

Appearance: Oval to angular cells with round nuclei and ABUNDANT cytoplasm

Cell Borders: Distinct cell junctions between attached cells

Name the Cell Type

• Size

• Clustering/clumping?

• Cellularity of FNA

• Morphological appearance

• Cell borders

Mesenchymal Cells

Size: Medium to large cells

Clustering: Usually individual cells

Cellularity: Exfoliate poorly so FNA contains very few cells → Scraping

Appearance: Spindle cells with cytoplasmic tails BUT may also appear plump

Cell Borders: Indistinct cell borders which fade into the background

Bound by bright pink ECM produced by the cells

What is required to to definitively diagnose a sarcoma (malignant mesenchymal neoplasia)?

Histology as the cells are indistinguishable from active young fibroblasts in granulation tissue

Round cells

• Size

• Clustering/clumping?

• Cellularity of FNA

• Morphological appearance

• Cell borders

Size: Small to medium cells (usually similar to RBCs)

Clustering: Usually single BUT may look like they are sticking

Cellularity: Exfoliate well on FNA

Appearance: Small, circular cells with round nuclei

Cell Borders: Distinct cytoplasmic borders

List the 5 (6) types of round cells

1. Lymphocytes

2. Plasma cells

3. Mast cells

4. Histiocytes

5. TVT

6. ± Melanocytes

Name the Round Cell

• Appearance

• 3 DDx

Lymphocyte

Appearance:

• Small, round cell

• Large nucleus which fills almost the entire cell

• Small rim of basophilic cytoplasm

DDx:

1. Normal lymphoid tissue

2. Reactive lymphoid tissue

3. Lymphoma

Name the Round Cell

• Appearance

• 2 DDx

Plasma Cell

Appearance:

• Eccentric nucleus (to one side)

• Moderate amount of DARKLY-stained basophilic cytoplasm (darker than lymphocytes)

• Often with pale area within cytoplasm = Golgi apparatus

DDx:

1. Plasma cell myeloma

2. Cutaneous plasmacytoma

Name the Round Cell

• Appearance

• DDx

Mast Cell

Appearance:

• Central to eccentric, round nucleus

• Abundant pale to clear cytoplasm

• PURPLE GRANULES

DDx: MCT

Name the Round Cell

• Appearance

• 2 DDx

Histiocyte

Appearance:

• "Fried egg" appearance

• Central to eccentric nucleus

• ABUNDANT clear to PALE basophilic cytoplasm

• Potentially with darker area of basophilia (opposite of plasma cell)

DDx:

1. Histiocytoma

2. Histiocytic sarcoma

Name the Round Cell

• Appearance

• Significance

Transmissible Venereal Tumour

Appearance: Histiocyte but vacuolated

Significance: NOT in NZ

• Usually found around the perineal area

Name the Round Cell

• Appearance

• DDx

Melanocyte

Appearance:

• Green/black granules in cytoplasm

• Moderate amount of basophilic cytoplasm

DDx: Melanoma (care with amelanotic melanoma = no granules)

Label the Following Round Cells

Histiocytes	Mast Cells	Melanocytes
TVT	Lymphocytes	Plasma Cells

FIVE: IS IT MALIGNANT OR BENIGN?

• Criteria of malignancy

  • 4 Whole cell features

  • 7 Nuclear features

• 3 Exceptions

Criteria of Malignancy: ≥3 criteria (more variation → more malignant)

• Whole Cell Features:

  1. Pleomorphism = Variability of the same cell type (anisocytosis, variable shape and high nucleus:cytoplasmic ratio)

  2. Disorganisation of cells within clusters

  3. Hypercellularity of smears (more exfoliating cells)

  4. Occasional very large cells

• Nuclear Features

  1. Macronuclei

  2. Anisokaryosis

  3. Multinucleation

  4. Macronucleoli (same size as RBC)

  5. Anisonucleoliosis (variation in nucleoli size and shape)

  6. Increased mitotic figures

  7. Abnormal mitotic figures

Exceptions:

1. Lymphoma (less variable than normal lymphoid tissue)

2. Thyroid carcinoma (uniform cells BUT malignant)

3. Apocrine gland adenocarcinoma (uniform cells but malignant)

Describe what is wrong with the following samples

Formalin = RBCs become green and background is murky	Splattergram = Material is expelled but NOT spread Cells are too close for examination	Squished Cells = Rough handling → Naked nuclei with nuclear debris
Slide Too Thick = Need to spread	Squished Cells = Rough handling → Naked nuclei with nuclear debris	Too Pink = Dip again into purple stain

Statement for:

1. Are there adequate cells present?

2. 2. Is the stain adequate?

3. Inflammatory vs. non-inflammatory (when it is non-inflammatory but there are neutrophils present)

Cells: Although there is blood contaminating the sample, there are sufficient intact cells which are spread out into a thin enough layer to obtain enough single cells to see cell definition

Staining: Yes. There is variation in colour and there is a clear difference between the nucleus, cytoplasm and background

Non-Inflammatory: Although there are neutrophils present, because they are the most abundant WBC in blood, and red cells are observed in the slide, the neutrophils likely they came from contamination of the sample with blood

Where to stop on the CSI-TM approach when round cell has been confirmed

Do not determine malignancy or benign as round cells are well-differentiated from each other and the type of cell provides a better indication for benign or malignant

List the only FOUR structures observed on an ear smear

1. Skin cells (squamous epithelial cells) ± melanin granules

2. Yeast (Malassezia spp.)

3. Bacteria (cocci and rods)

4. Neutrophils

Label the Following Image

White Arrow = Melanin pigment within keratocytes

• Common in dark-skinned animals

Red Arrow = Bacteria (cocci)

Green Arrow = Degenerate neutrophils

Label the Following Image

Grey Arrow = Keratin bars/scrolls (rolled up keratinised epithelial cells)

Red Arrow = Bacteria

Label the Following Image

Grey Arrow = Neutrophils

Red Arrow = Cocci

Green Arrow = Rods

Label the Following Image

Yellow Arrow = Melanin granules

Red Arrows = Yeast

Large dark blue structures = Epithelial cells

How to differentiate yeast from bacteria on ear smear

Yeasts are MUCH larger than bacteria 40x = easily see yeast while bacterial presence is questionable

• 100x = BOTH (yeasts are obvious and bacteria more clearly visible)

• Yeast = Footprints

Effusion

• Definition

• Normal

Definition: Accumulation of fluid in the body cavity produced by mesothelial cells of the visceral and parietal serosal lining

Normal: <1mL for lubrication

What is Starling's law?

Defines the retention of fluid in vessels which is determined by FPnet (net filtration pressure)

• FPnet = HSP - COP (hydrostatic pressure - colloid osmotic pressure)

Describe the 5 steps of fluid sample collection

1. Clip site and surgically prep area for aseptic technique

2. FNA with 18 - 22G needle

3. Place fluid into an EDTA tube to prevent clot formation

4. Prepare fluid sample that is NOT from the EDTA tube (EDTA is bactericidal)

5. Make smears like a blood smear OR stop prep OR centrifuge to remove supernatant

4 Step of preliminary fluid analysis after sample collection

1. Assess gross appearance before and after centrifuge (colour and turbidity)

2. Cytology and cell counts to assess cellularity

3. TP with refractometer (centrifuge to measure protein of supernatant first)

4. ± Culture and sensitivity

What are these? What is their significance?

Reactive Mesothelial Cells = Mesothelial cells lining the pleural, peritoneum and pericardium become reactive (produce more fluid), → Plump up and divide and hence often have:

1. TWO nuclei but are NOT neoplastic

2. Pink fringe/tutu

Difficult to differentiate from neoplastic mesothelial cells

Fill out the Following Table on Classifications of Effusions

Feature	Transudate	Modified Transudate	Exudates
Colour and consistency
TP
Nucleated cell count (TNCC)
Specific gravity
Cells
Mechanism
Cause
Clot

Feature	Transudate	Modified Transudate	Exudates
Colour and consistency	Clear and watery	Vague intermediate	Turbid (cloudy) and thick
TP	Low (<25g/L)	Moderate (25 - 50 - 70g/L)	High (>30g/L)
Nucleated cell count (TNCC)	Low (<0.5 x 10^9/L)	Moderate (0.5 - 7 x 10^9/L)	High (>3 x 10^9/L)
Specific gravity	Low (<1.017)	Moderate (1.017 - 1.025)	High (>1.025)
Cells	Normal cell (mainly mononuclear, occasional non-degenerate neutrophils ± mesothelial cells)	Reactive mesothelial cells, macrophages, non-degenerate neutrophils, lymphocytes	Septic: Large number of degenerate neutrophils ± bacteria Often sediments on centrifugation Non-septic (pancreatitis, bladder rupture): Non-degenerate neutrophils ± neoplastic cells exfoliate into fluid
Mechanism	Low COP or increased HP (non-inflammatory)	Increased HP or impaired lymphatic drainage Cells and protein added to transudate over time due to mild inflammation from fluid which irritates the mesothelial cells which become hyperplastic and slough into fluid (some neoplastic effusions)	Increased vascular permeability
Cause	Severe hypoalbuminaemia (liver failure) and early heart failure	CHF Neoplasia Organ torsion or herniation Pulmonary thrombosis Heartworm disease Liver disease (portal hypertension) Pericardial effusion Diaphragmatic hernia	Inflammatory ± septic (bacteria)
Clot	No	Mixed	Yes (high fibrinogen)

List 6 effusions which do not fit into the categories well

1. Haemorrhage (frank blood)

2. Chylous effusions

3. Ruptured abdominal organs (eg. uroperitoneum or bile peritonitis)

4. Equine abdominal paracentesis

5. Feline infectious peritonitis

6. Neoplastic effusion

Haemorrhagic Effusion

• Appearance

• 2 alternative DDx and differentiation

• Types of cells present >few hours old and greater than 2 days old

Appearance: Red or serosanguinous colour → Brown

DDx: ALSO due to contamination at sampling or accidental splenic aspiration (would have platelets and clumping)

Cells:

• < Few hours = Macrophages with RBCs in cytoplasm

• >2d = Macrophages with haemosiderin

Chylous Effusion

• Appearance

• MoA

• Typical location

• Cells present (4)

• 4 DDx

• 3 Diagnostic methods

Appearance: Milky white effusion (may be pink with blood)

MoA: Leakage of lymphatic vessels carrying fat

Location: Thorax #1

Cells:

1. Small lymphocytes

2. Macrophages (with lipid vacuoles)

3. Neutrophils

4. Fat droplets

DDx:

1. Heart disease in cats (cardiomegaly prevents emptying of thoracic ducts)

2. Neoplasia (eg. lymphoma or thymoma)

3. Lymphatic rupture

4. Lung torsion

Diagnosis:

1. Centrifuge sample (remains white if chylous)

2. Measure TAG content compared to serum (chylous TAG > 2 - 3x serum TAG)

3. Refrigerate sample overnight (fat rises to top and solidifies)

What does presence of plant material in paracentesis indicate in a horse?

Ruptured GIT

CASE 1: 1yr X-breed

History: Dog presented with pendulous abdomen. Abdominal radiology confirmed fluid in the abdomen

Question: CSI-TM approach and provide and interpretation/diagnosis

CSI-TM:

1. Useful cells present? Yes (lots of intact cells present, thinly enough spread to see cell definition and not just blood)

2. Adequate staining? Yes (can see cytoplasm vs. nucleus and background is clear)

3. Inflammatory vs. non-inflammatory? Inflammatory

   • Lots of neutrophils, some degenerate

   • Occasional intracellular bacteria (rod-shaped)

Interpretation: Septic, suppurative peritonitis

CASE 2: 6yr ME GSD

History: Presented for scooting and difficulty defaecating

PE: Mass on rectal palpation

Question: CSI-TM approach and provide and interpretation/diagnosis

CSI-TM:

1. Useful cells? Yes

   • Lots of intact cells that are thin enough to see cell definition

2. Adequate staining? Yes (can see difference between nucleus and background)

3. Inflammatory or non-inflammatory? Non-inflammatory (no neutrophils and macrophages)

4. Cell type? Epithelial (cell clustering and abundant cytoplasm)

5. Malignant or benign? Benign

Interpretation: Benign epithelial mass (perianal adenoma)

CASE 3: 14yr DSH cat

History: Multiple small (<1cm) skin masses over entire body

PE: Splenomegaly and hepatomegaly

Question: CSI-TM approach and provide and interpretation/diagnosis

CSI-TM:

1. Useful cells? Yes

2. Adequate staining? Yes

3. Inflammatory or non-inflammatory? Non-inflammatory

   • Neutrophils present due to blood contamination (RBCs present)

4. Cell type? Round cells (mast cells)

Interpretation: Mast cell tumour

CASE 4: 9yr DSH

History: Cat presented for coughing and lethargy

PE: Pleural fluid found

Question: CSI-TM approach and provide and interpretation/diagnosis

CSI-TM:

1. Useful cells? Yes

2. Adequate staining? Yes

3. Inflammatory or non-inflammatory? Non-inflammatory

4. Cell type? Epithelial cells (clustering)

   • Signet ring = Epithelial cells swell and have a chunk at the top

5. Malignant or benign? Malignant (highly variable, lots of balloon cells, anisocytosis, anisokaryosis = variable nuclear cells, signet rings)

Interpretation: Carcinoma

CASE 5: 9yr Rottweiler

History: Presented 3-legged lame (non-weight bearing) on right forelimb

PE: Extreme pain of right shoulder

Radiographs: Lysis of proximal humerus

Question: CSI-TM approach and provide and interpretation/diagnosis

CSI-TM:

1. Useful cells? Yes

2. Adequate staining? Yes

3. Inflammatory or non-inflammatory? Non-inflammatory

4. Cell type? Mesenchymal (tails, not clustering, fairly large)

5. Malignant or benign? Malignant (highly variable, lots of balloon cells, anisocytosis, macronucleoli, angular nucleoli)

Interpretation: Sarcoma (osteosarcoma)

CASE 6: 11yr cross-breed dog

History: Large abdominal wall mass

Question: CSI-TM approach and provide and interpretation/diagnosis

CSI-TM:

1. Useful cells? Yes

2. Adequate staining? Yes

3. Inflammatory or non-inflammatory? Both

   • Neutrophils and occasionally macrophages

4. Cell type? Epithelial (clumping)

5. Malignant or benign? Malignant (highly variable, balloon cells, anisocytosis and anisokaryosis)

Interpretation: Carcinoma with inflammation

CASE 7: Horse with colic

PE: Abdominal fluid aspirated and shown pre- and post-centrifugation

Question: CSI-TM approach and provide and interpretation/diagnosis

CSI-TM:

1. Useful cells? Yes

   • Grass in peritoneal cavity = Intestinal rupture

2. Adequate staining? Yes (not a lot of variation in colour, but only single cell type)

3. Inflammatory or non-inflammatory? Inflammatory

   • Neutrophils, some degenerate

   • Some intracellular bacteria = septic

Interpretation: Septic peritonitis

CASE 8: 11yr Cat

History: Lethargy and an extended abdomen

PE: Ascites

Question: CSI-TM approach

CSI-TM:

1. Useful cells? Yes

2. Adequate staining? Yes

3. Inflammatory or non-inflammatory? Non-inflammatory

4. Cell type? Epithelial (clumping)

   • LARGE compared to neutrophils (round cells would be similar size to leukocytes

5. Malignant or benign? Malignant

   • Anisokaryosis

   • Anisocytosis

   • Anisonucleoliosis (macronucleoli as big as RBCs)

CASE 9: 7m Burmese

History: Extreme lethargy

PE: Abdominal fluid

Question: CSI-TM approach and provide and interpretation/diagnosis

CSI-TM:

1. Useful cells? Yes

2. Adequate staining? Yes

3. Inflammatory or non-inflammatory? Inflammatory

   • Neutrophils with some macrophages

   • Organisms? None

   • High protein fluid = Purple background

   • Fingernail clippings = Protein folds

Interpretation: Mixed inflammation in abdomen (FIP)

CASE 10: 13yr MC Corgi

History: Skin mass on flank

Question: CSI-TM approach and provide and interpretation/diagnosis

CSI-TM:

1. Useful cells? Yes

2. Adequate staining? Yes

3. Inflammatory or non-inflammatory? Non-inflammatory

4. Cell type? Round cells = Plasma cells

Interpretation: Plasmacytoma

CASE 11: 8yr FS Labrador

History: Skin mass on HL

Question: CSI-TM approach and provide and interpretation/diagnosis

CSI-TM:

1. Useful cells? Yes

2. Adequate staining? Yes

3. Inflammatory or non-inflammatory? Non-inflammatory with single ruptured eosinophil (must be >10% to be eosinophilic inflammation)

4. Cell type? Round cell = Mast cell

Interpretation: Mast cell tumour

CASE 12: 4yr Rottweiler

History: Swollen stifle and dog was in a fight a few days ago

Question: CSI-TM approach and provide and interpretation/diagnosis

CSI-TM:

1. Useful cells? Yes

2. Adequate staining? Yes

3. Inflammatory or non-inflammatory? Inflammatory

   • Unknown if bacteria present (requires 100x)

   • Neutrophils in lines = Wind rowing (joints have highly proteinaceous background due to high viscosity)

Interpretation: Suppurative arthritis (immune-mediated OR septic)

CASE 13: 10yr Dalmatian

History: Skin mass in preputial area

Question: CSI-TM approach and provide and interpretation/diagnosis

CSI-TM:

1. Useful cells? Yes

2. Adequate staining? Yes

3. Inflammatory or non-inflammatory? Non-inflammatory

4. Cell type? Round cell = Melanocyte

Interpretation: Melanoma

CASE 14:

History: Skin mass on lateral wall of abdomen, near thigh

Question: CSI-TM approach and provide and interpretation/diagnosis

CSI-TM:

1. Useful cells? Yes

2. Adequate staining? Yes

3. Inflammatory or non-inflammatory? Non-inflammatory (neutrophils = blood contamination)

4. Cell type? Mesenchymal

5. Malignant or benign? Malignant (anisocytosis, anisokaryosis, anisonucleoliosis)

Interpretation: Sarcoma

CASE 15: Dog

History: Abdominal mass

Question: CSI-TM approach and provide and interpretation/diagnosis

CSI-TM:

1. Useful cells? Yes

2. Adequate staining? Yes

3. Inflammatory or non-inflammatory? Non-inflammatory

4. Cell type? Epithelial

5. Malignant or benign? Malignant (anisocytosis, abnormal mitotic figure, anisokaryosis, anisonucleoliosis)

Interpretation: Carcinoma

CASE 16: Cat

History: Fluid in thorax

Question: CSI-TM approach and provide and interpretation/diagnosis

CSI-TM:

1. Useful cells? Yes

2. Adequate staining? Yes

3. Inflammatory or non-inflammatory? Inflammatory with intracellular rods

Interpretation: Septic pyothorax

CASE 17: 1.5yr Poodle

History: Mass on muzzle

Question: CSI-TM approach and provide and interpretation/diagnosis

CSI-TM:

1. Useful cells? Yes

2. Adequate staining? Yes

3. Inflammatory or non-inflammatory? Non-inflammatory

4. Cell type? Round cell = Histiocytes

Interpretation: Histiocytoma

CASE 18: Dog

History: Intra-abdominal mass

Question: CSI-TM approach and provide and interpretation/diagnosis

CSI-TM:

1. Useful cells? Yes

2. Adequate staining? Yes

3. Inflammatory or non-inflammatory? Non-inflammatory

4. Cell type? Round cell = Lymphocytes

Interpretation: Lymphoma

CASE 19: Cat

History: Fluid in thorax

Question: CSI-TM approach and provide and interpretation/diagnosis

CSI-TM:

1. Useful cells? Yes

2. Adequate staining? Yes

3. Inflammatory or non-inflammatory? Inflammatory with intracellular rods

Interpretation: Septic pyothorax

3 Indications of fresh/frozen samples for PM

Infectious/toxic causes

1. Bacterial culture

2. PCR

3. Toxicology

Direct vs. Indirect Testing

• Definition

• 3 Examples of each

Direct Testing = Identifies presence of agent itself

Examples:

1. Bacterial or fungal culture → Promotes growth/replication of infectious agent for identifications

2. PCR → Extract, amplify and sequence DNA of infectious agent from sample

3. Serology Ag test → Add Ab to sample which binds with the Ag to form an Ag-Ab complex which can be visualised

Indirect Testing = Identifies animal's response to agent (i.e. Ab) vs. agent itself

Examples:

1. IFA

2. ELISA

3. MAT

Serology

• Definition

• MoA

• 4 Types

  • Qualitative vs. quantitative

  • MoA

Definition: Examining serum/other bodily fluids (milk, CSF) for presence of Ab/Ag of an infectious agent (occasionally of a hormone, enzyme like pancreatic lipase or auto-Ab)

MoA: Ag binds specific Ab → Ab-Ag complex formed → Indicators bind complex to visualise reaction and ID positive result

• Indicators: Coloured indicator dyes, fluorescent markers or agglutinating particles (eg. latex)

• Intensity of reaction is proportional to amount of Ab/Ag present in sample

Types:

1. ELISA (Enzyme-linked immunosorbent assay) = Qualitative

   • Ag (plates/wells coated with Ab which Ag in sample binds to)

   • Ab (plates/wells coated with Ag for Ab in sample to bind to)

   • 2nd Ab added after sample which is linked to a chromogenic indicator molecule → Sandwich

   • Positive result: Colour change

     

2. RIM (Rapid immuno-migration tests) = Qualitative

   • Type of ELISA test in cassette form (aka. SNAP or POC tests)

   • eg. FeLV Ag, parvovirus Ag and FIV Ab

     

3. Agglutination tests (LAT and MAT) = Qualitative (titres)

   • Use the tendency of Ag to clump together (agglutinate) in the presence of specific Ab in suspension

   • LAT = Latex particles bind Ag-Ab complexes when present → Detectable mat which floats to surface (eg. Toxoplasma gondii Ab)

   • MAT = Examine with dark-field microscopy for Ab-Ag complex formation and agglutination (eg. Leptospiral Ab)

     

4. IFA and TIA (Immunofluorescence assay and turbidimetric immunoassay) = Qualitative (titres)

   • eg. Failure of passive transfer in foals

Titres

• Definition

• 2 Example tests that measure titres

• Paired titres

Definition: Way to express concentration (quantity) of particular Ab in sample

• Determined by serially diluting sample and testing each → Final titre = Highest dilution that still produces positive test reaction

• Higher denominator for final titre → More Ab present in sample (eg. 1/512 is a stronger positive result than 1/32 → 1/512 more likely to indicate active of current infection)

Tests:

1. Agglutination tests (LAT and MAT)

2. IFA

Paired Titres: TWO samples taken 2 - 4 weeks apart to determine if recent/current infection is present (4-fold rise in Ab titres)

2 Disease vaccinations that influences SNAP results

1. Cats vaccinated against FIV → Positive Ab result

2. Dogs vaccinated against parvovirus in last 7 - 10 days → Weak positive Ag result

5 Causes of low titre (<1/800) for Leptospirosis

1. Recent infection (titre still rising)

2. Recent vaccination

3. Chronic infection (subclinical)

4. Convalescent titre (dropping after previous infection)

5. Maternal antibody in calves → Distinguish from recent infection with paired titres

Serum sample from with with 1/200 titre for Leptospira hardjo and 1/1600 titre for Leptospira pomona Which serovar is the likely cause of leptospirosis?

L. pomona is the likely cause of current infection and L. hardjo titre likely due to previous vaccination

PCR

• 5 Advantages

• 3 Disadvantages

• 4 Samples to collect

+ve:

1. Small sample volumes (often swab sufficient) necessary (vs. > 1 - 2mL of blood for serology)

2. High Sn and Sp

3. Infectious agent does NOT need to be alive (VS. bacterial/fungal culture)

4. Can detect early in disease course (do not have to wait for Ab to rise)

5. Cost effective screening for some diseases and performed on samples pooled from multiple animals

-ve:

1. May need multiple PCR tests for different infectious agents to make diagnosis → $$

2. Quality of sample, primers and protocol influences Sp and Sn

3. False positive possible (eg. tiny remnant of Toxoplasma in sheep sample → positive PCR despite NOT being cause of abortion)

Samples: Depends on disease being tested (check with lab first)

1. Sterile swabs

2. Fluids samples

   • MCF = EDTA vs. BVD = Serum

3. Faeces (GIT disease)

4. Stomach contents of aborted foetus

What type of virus is tested on FIP PCR?

FeCV (some tests distinguish between FeCV and FIPV, but the normal FeCV should not be present in effusion anyway)

Parvovirus Diagnosis

• Test

• 3 Disadvantages

Test: SNAP RIM for Ag in faeces

-ve:

1. False positive due to recent vaccination with modified live vaccine in last 7 - 10 days

2. False negative due to transient shedding

3. False negative due to faecal Ab present binds the virus

FPT in Foals

• 2 Tests (+ sample)

• When should blood be collected?

Tests: Horses do NOT have high levels of GGT in colostrum

1. IgG (Ab) TIA test at diagnostic lab = Serum

   • Quantitative and higher DSe

2. SNAP IgG (Ab) "foal side" test = Serum

   • Results within 7 minutes

   • Qualitative result (<400, 400 - 800 or >800mg/dL = Failure, partial failure and adequate passive transfer respectively)

Collection: 18 - 24hr post-birth

BVDV Testing

• 6 Tests (+ sample)

• First BVD test to perform on a dairy herd + sample submitted

  • Results

  • Follow-up test

Tests:

1. Pooled Ab ELISA = Bulk milk tank/serum

2. Individual Ab ELISA = Individual milk/serum

3. Individual Ag ELISA = Serum or ear notch (ear notch only <35 days as maternal Ab from colostrum neutralise viral Ag in blood)

4. Individual Ag RIM/SNAP = Ear notch (2 - 3mm tissue)

5. Pooled PCR = Milk or serum

6. Individual PCR = Serum or ear notch

1st Test: Bulk milk tank ELISA Ab (or pooled serum) → Define BVD status of herd through S/P ratio

• S/P > 0.75 = High number of seropositive cows indicates current or recent exposure to BVD due to presence of PI animal → Individual Ag ELISA for PI hunt

• 0.17 < S/P < 0.75 = Presence of transiently infected (TI) animals rather than PI animals → Assess bulk tank milk for BVD Ag to ID any unidentified PI animals in the milking herd for PI hunt

  1. Cows infected with PI animal over fence line

  2. Contact with PI animal that has since died/been culled

  3. PI animal recently introduced and not yet had time to infect many other animals

  4. PI present but extensive grazing results in low contact rates between animals

• S/P < 0.17 = No PI animal

2 Test options for screening young stock for BVD for PI animals

1. Direct testing (serum or ear notch) for BVD virus with PCR or ELISA Ag +ve result = PI or TI → Retest 3 - 4 weeks later TWO +ve results → PI

2. Indirect testing (serum) for BVD Ab with ELISA ONLY recommended in unvaccinated animals with low chance of PI animal in the group as it relies on the fact that in herds with PI, many have high Ab titre

6 Reasons for a false negative test result

1. Ag tests in late stages of infection (eg. intermittent and reduced shedding as disease progresses)

2. Ab tests in early stages of infection (prior to effective seroconversion)

3. Immunocomplexing (endogenous Ab binds Ag which prevents it from being free to bind the test reagent Ab)

4. Wrong/bad sample tested (Ag or Ab in sample becomes degraded and will not react)

5. Test not performed correctly

6. Wrong test

2 Reasons for a false positive test result

1. Ab (and some Ag) tests with recent vaccination (many tests cannot distinguish between Ab produced in response to vaccination vs. infection)

2. PCR tests in general (very sensitive eg. using sample needle on multiple animals)

What steps would you recommend for a farm dog with suspected leptospirosis presenting acutely?

Given the acute presentation, I would try for a PCR on a urine sample first to detect Leptospirosis Ag. Ab are unlikely present in serum as there has not been sufficient time for seroconversion. PCR is not specific to a serovar but the disadvantage is false negatives due to intermittent shedding. If the test is negative, but I still highly suspect Leptospirosis, I would then do a serology for Ab with MAT. Because I must select a specific serovar, I would start with Hardjo and Pomona as this is a farm dog, and go from here (save owner cost). These tests must be repeated in 2 - 4 weeks to show a rising titre (>4-fold increase). As such, I would treat for leptospirosis while awaiting the 2 - 4 weeks to pass.

3 Potential causes for Leptospira copenhagenii titre of 1/50

1. Not infected (or recent vaccination)

2. Infected with different serovar

3. Infected with copenhagenii, but insufficient time for seroconversion → False negative