Purdue University

School of Veterinary Medicine

and

IDEXX Laboratories

2006 Clinical Pathology

Symposium:
Cytology
Effusions by
Craig Thompson DVM, DACVP
Clinical Assistant Professor
•Fat
•Food
•Fetus
•Flatus
•Feces
•Foreign
Body
•Fluid
•Fat
•Food
•Fetus
•Flatus
•Feces
•Foreign
Body

•*Fluid*
 Anatomy of Serous
Cavities
ƒ Peritoneal
– Mesothelium lined potential space
– Hiatuses
ƒ Esophageal
ƒ Vena caval
ƒ Aortic
– High rate of fluid turnover
ƒ Sternal lymph nodes: 4/5
ƒ Omental lymph nodes: 1/5
 Anatomy of Serous
Cavities
ƒ Peritoneal
– Parietal
ƒ Abdominal
ƒ Pelvic
ƒ Scrotal
– Visceral
ƒ Covers abdominal viscera
ƒ Higher capillary pressure predisposes cavity
to fluid accumulations
 Anatomy of Serous
Cavities
ƒ Pleural
– Mesothelium lined potential space
ƒ Lung movements
ƒ Cardiac motion
ƒ Transmission of forces
– Visceral
ƒ Covers lungs
– Parietal
ƒ Costal, diaphragmatic and mediastinal
 Anatomy of Serous
Cavities
ƒ Pleural
–Mediastinum
ƒ Separates left and right hemi-thoraces
ƒ Complete- Bovid, goats, pigs, young
animals
ƒ Fenestrated- Dogs, cats, horses and
sheep
 Anatomy of Serous
Cavities
ƒ Pericardial
–Mesothelium lined space
ƒ Cardiac motion
ƒ May serve to isolate heart from thorax
ƒ Fluid accumulation rapidly detrimental
ƒ Superfluous: Removal or fenestration
yields no major side effects
 Effusions
Physiology

• Plasma filtrate
• Multiple factors involved
• Species and cavity differences
• Starlings forces
• Large amount of daily turnover, yet stable,
small amount of fluid present in any given
cavity at any time
 Effusions
Pathophysiology

• Disturbance in Starlings forces

• Hypoproteinemia, especially albumin
• PLE, PLN, Liver failure
• Increased hydrostatic pressure
• Heart failure, portal hypertension
• Increased vascular permeability
• Vasculitis, response to mediators
• Decreased lymphatic drainage
• Neoplasia, inflammation
 Effusions
Pathophysiology

• Other sources
• Hemorrhage
• Compromise of the urinary tract
• Neoplastic cells
• Ruptured intestine
• Iatrogenic
 Cytology of Effusions
Clinical Utility

• Help rule in or out differential diagnoses

derived from other modalities
• Minimally invasive
• Pericardial > Pleural > Peritoneal
• Short learning curve
• Diagnostic +/- therapeutic
 Cytology of Effusions
Sample Collection
• Abdominocentesis
• Usually indicated due to physical
examination or diagnostic imaging findings
• “Dry” taps
• Avoid masses, including bladder
• Surgical preparation
• Choice of weapons to obtain sample
• Gravity vs. negative pressure
• Observe sample as it is collected
 Cytology of Effusions
Sample Collection
• Thoracocentesis
• Usually indicated due to physical
examination or diagnostic imaging findings
• Surgical preparation
• Cranial to rib
• Maintain negative pressure
• Amount removed
- Diagnostic
- Therapeutic
 Cytology of Effusions
Sample Collection

• Pericardiocentesis
• Usually indicated due to physical
examination, diagnostic imaging or
electrocardiographic findings
• Surgical preparation- Right 3rd to 8th ICS
• Optional ECG monitoring in case of
ventricular puncture
 Cytology of Effusions
Sample Collection

• Diagnostic Peritoneal Lavage (DPL)

• Usually indicated after repeated “dry” taps
• Introduce catheter and wait
• 20 ml/kg warm, isotonic crystalloid
• Agitate abdomen
ÌSample will be undeterminably dilute
• Can leave catheter in place for therapy
 Cytology of Effusions
Sample Handling

• Tube selection
• EDTA / Lavender top
- Ideal for cell morphology, staining, etc.
- TP can be determined by refractometry
- Cells will vacuolate with time
- NOT suitable for bacterial cultures
 Cytology of Effusions
Sample Handling

• Tube selection
• No Additive / Red top
- Used for biochemical assays
- NOT suitable for cell enumeration
- Can be used for culture
- Serum Separator Tubes (STT) should
never be used for fluid collection
• Heparin / Green top
- Used for biochemical assays
 Cytology of Effusions
Sample Handling

• Tube selection
• Culturettes / Culture Tubes
- Aerobic bacterial
- Anaerobic bacterial
- Fungal
- Other agents
- Can use syringe w/ needle
ÌCall ahead
 Cytology of Effusions
Sample Handling

• Delay in processing
• Make fresh smears- Reference point
- Direct
- Concentrated
- Line smears
- Label
 Cytology of Effusions
Sample Handling
• Shipping
• Sample on ice
• Overnight
• Slides in slide
transporters
 Cytology of Effusions
Sample Preparation

• Direct smear
• Clean, new slides
• High nucleated cellularity
• Place drop at end of slide
• Back up spreader slide
• One smooth, straight stroke
• Label appropriately: “Str”
 Cytology of Effusions
Sample Preparation
• Line technique
• Clean, new slides
• Moderate nucleated cellularity
• Place drop at end of slide
• Back up spreader slide
• One smooth, straight stroke
ÌStop short of “feathering”
• Label appropriately
 Cytology of Effusions
Sample Preparation

• Concentrated smear
• Clean, new slides
• Low nucleated cellularity
• Centrifuge aliquot
• Pour off supernatant
• Re-suspend (0.5ml)
• Place drop at end of slide
• Back up spreader slide
• One smooth, straight stroke
• Label appropriately: “Sed”
 Cytology of Effusions
Sample Preparation

• Cytocentrifuge preparation
• For very low cellularity
• Not readily available
• Low speed centrifuge
• Cells spun directly onto slide
• Supernatant wicked away
* Can use gravity apparatus
 Cytology of Effusions
Stains

• Diff-Quik • Gram
• Polychromatic • Categorize bacteria
• Good overall detail • Poor cellular detail
• Art to consistency * Control slide
• Wright’s • New Methylene Blue
• Excellent detail • Good vs. hemodilution
• Automated • Marginal cellular detail
Fibrosarcoma. Diff-Quik. 100x Osteosarcoma. Modified Wright’s. 100x
Pancreatic abscess. Modified Wright’s. 100x Pancreatic abscess. Gram. 100x
 Cytology of Effusions
Gross Examination
• Color: Straight v. Supernatant
• Particulate
- Cells
- Fibrin clumps
- Debris
- Ingesta
• Non-particulate
- Pigments: Hemoglobin, myoglobin, bilirubin
- Lipid
 Cytology of Effusions
Gross Examination
• Colors:
• Red – Pink: RBCs and/or hemoglobin
• Bright red: Whole blood
• Reddish brown: Old hemorrhage
• Muddy or port wine: Necrosis
• Brown: Melanin
• Dark green: Bile
• Bright green: Enterocentesis
• Light green: Eosinophils
• White: Lipid or WBCs
 Cytology of Effusions
Gross Examination
• Clarity: Straight v. Supernatant
• Particulate
- Cells
- Fibrin clumps
- Debris
- Ingesta
• Non-particulate
- Pigments: Hemoglobin, myoglobin, bilirubin
- Lipid
 Cytology of Effusions
Gross Examination

• Odor
• Foul- Anaerobic infection
• Enterocentesis: Fermented
• Uroabdomen: Ammonia
 Cytology of Effusions
Chemical Analysis

• Total Protein
• Used to classify effusions
• Often use refractometer on supernatant
• Can use chemistry analyzer
• Others
Triglycerides Lactate
Cholesterol pH
Glucose Creatinine
 Cytology of Effusions
Cell Quantification

• Total Nucleated Cell Count (TNCC)

• Used to classify effusions
• Unopette system
• Automated cell counter
• Erythrocytes
• Automated cell counters often give RBC
• Microhematocrit PCV just as good
Cytology of Effusions
Classification

Class TP g/dl TNCC /μl

Transudate <2.5 <1,000
(<10,000 Eq)

Modified 2.5-7.5 1,000 – 7,000

Transudate
Exudate >3.0 >7,000
 Cytology of Effusions
Microscopic Examination

• Erythrocytes
• Yes or No
• Don’t over interpret
• Look for evidence of hemorrhage
• If in doubt, phlebotomy
Mycoplasma haemocanis. Modified Wright’s. 100x

Canine poikilocytes. Modified Wright’s. 100x

 Cytology of Effusions
Microscopic Examination

• Mesothelial cells
• Simple squamous cells
• Commonly seen in all classes of effusions
• Typically grouped with macrophages as
“Large mononuclear cells”
 Cytology of Effusions
Microscopic Examination

• Mesothelial cells
• Non-Reactive
- Small to medium sized
- Singlets and small to medium sized clusters
- Single round nucleus set in moderate to large
amount of smooth periwinkle colored cytoplasm
- Corona occasionally seen
- Finely granular chromatin pattern
- Rare to occasional small nucleoli
Canine. Non-reactive mesothelial cells
Modified Wright’s. 100x
 Cytology of Effusions
Microscopic Examination
• Mesothelial cells
• Reactive- Secondary to many things
- Small to large sized
- Singlets and small to medium sized clusters
- One to several, round to markedly pleomorphic
nuclei set in scant to large amount of deep purple
colored cytoplasm
- Occasionally contain vacuoles
- Coarse to clumped chromatin pattern
- One to several, markedly pleomorphic nucleoli
Canine. Reactive mesothelial cells
Modified Wright’s. 100x
Canine. Reactive mesothelial cells. Modified Wright’s. 100x
 Cytology of Effusions
Microscopic Examination

• Macrophages- Non-Activated
• Small to medium in size
• Always discrete
• Single round to indented nucleus set in
moderate to large amount of smooth blue
cytoplasm
• Cell margins occasionally ruffled
• Finely stippled chromatin pattern
Canine. Non-activated macrophages
Modified Wright’s. 100x
Canine. Non-activated macrophages (arrows), Activated
macrophage with phagocytized debris (center)
Modified Wright’s. 100x
 Cytology of Effusions
Microscopic Examination

• Macrophages- Activated
• Medium to large sized
• Always discrete
• One to a few, indented to pleomorphic nuclei set
in scant to large amount of heavily vacuolated
cytoplasm
• Occasionally contain phagocytized cells / debris
Canine. Reactive mesothelial cells (arrows) with several activated
macrophages
Modified Wright’s. 100x
 Cytology of Effusions
Microscopic Examination

• Lymphocytes
• Mature lymphocytes seen in effusions
• Morphologically similar to peripheral blood
• Occasional reactive cells seen
• Slightly larger with more, and darker cytoplasm
• Perinuclear clearing
• Large numbers of immature cells consistent
with lymphomatous effusion
Normal equine lymphocyte. Modified Wright’s. 100x

Reactive lymphocytes. Modified Wright’s. 100x

Neoplastic lymphocytes. Modified Wright’s. 100x
 Cytology of Effusions
Microscopic Examination
• Neutrophils
• Frequently seen with contamination as well
as true in vivo change
• 1:400 ratio for neutrophil : RBC
• Look for other signs of blood contamination
and lack of signs of hemorrhage
• Always assess neutrophils and sub-classify
- Non-degenerate
- Degenerate
- Aged
 Cytology of Effusions
Microscopic Examination

• Neutrophils- Non-degenerate
• Look exactly as in circulation
• Condensed, dark purple chromatin pattern
• Crisp nuclear margins
• 4-5 nuclear lobes
Canine non-degenerate
neutrophils
Modified Wright’s. 100x
 Cytology of Effusions
Microscopic Examination
• Neutrophils- Degenerate
• Some agent is killing the neutrophils in situ
- Bacterial toxins
- Bile
- Pancreatitis mediators
- Air
• Toxic changes- Cytoplasmic, in bone marrow
• Degenerate changes- Nuclear, occur
generally in the tissues
 Cytology of Effusions
Microscopic Examination
• Karyolysis- Changes occur with intact
cytoplasm
• Nucleus diffusely swells
• Chromatin stains less intensely
• Nucleus can hyalinize and look glassy
• Nuclear margins becomes indistinct
• Complete dissolution of nucleus
• Karyorrhexis- Fragmentation and condensation
of nucleus into multiple hyperchromic spheres
Degenerate neutrophils
(clockwise from upper left):
Non-degenerate, slightly swollen,
more swelling, pale staining,
swollen and somewhat glassy
chromatin
Karyorrhexis (arrow) and mild to moderate karyolysis
Canine. Modified Wright’s. 100x
 Cytology of Effusions
Microscopic Examination
• Neutrophils- Aged
• Neutrophil tissue life is hours
• Do not re-enter the circulation
• Benign environment or low grade toxin
• Variants of apoptosis
- Hypersegmentation- >5 lobes, thin connections
- Pyknosis- Nuclear and cellular condensation with
some fragmentation of nucleus
- Leukocytophagia- Large mononuclear cells
phagocytize
Hypersegmented
neutrophils. Canine
left, equine above
 Pyknotic cell and nucleus (arrow)
Modified Wright’s. 100x

Neutrophil with a pyknotic nucleus (arrow)

Modified Wright’s. 100x
Leukophagia (center). Canine. Modified Wright’s. 100x
 Cytology of Effusions
Microscopic Examination

• Other Cells
• Eosinophils
- Similar to those seen in circulation
- Off staining granules
- Hyposegmentation
• Mast Cells
- Similar to those seen in circulation
 Cytology of Effusions
Microscopic Examination

• Non-cellular elements
• Glove powder
- Polygonal
- Crystalline
- Refractile

• Lysed cells
- Must have intact cytoplasm and nucleus to
evaluate
 Cytology of Effusions
Microscopic Examination

• Non-cellular elements
• Ultrasound gel
- Granular
- Amorphous
- Bright pink
 Cases
Pure Transudate

• Protein- <2.5 g/dl

• Cells
- Large mononuclear cells (non-reactive, non-activated)
- Lymphocytes, non-reactive
- Non-degenerate neutrophils
• Features- Neutrophil hypersegmentation, pyknosis
• Ancillary tests- None
 “Teddy”
Canine- 6mo. M
Color: Straw
Clarity: Clear
S Color: Straw
S Clarity: Clear
TP: 2.3 g/dl
TNCC: 750/ μl
RBC: <30,000/μl

Canine peritoneum
Cytocentrifuge preparation
Modified Wright’s 100x
 Canine 7 YO SF
Color: Straw
Clarity: Clear
S Color: Straw
S Clarity: Clear
TP 0.5 mg/dl
TNCC: 140/ μl
RBC: <30,000/ μl

Canine pleura
Concentrated smear
Modified Wright’s 100x
 Cases
Septic Exudate
• Protein- Usually >3.5 g/dl, not uncommonly >5.0 g/dl
• Cells- TNCC can be >200,000/μl
- Predominantly non-degenerate to markedly degenerate neutrophils
- Activated and phagocytic macrophages frequently found
- Markedly reactive mesothelial cells
• Features
- Locate and identify organism
• Ancillary tests
- Culture and sensitivity are indicate if organisms seen within cells;
however, degenerate neutrophils and elevated TNCC raise suspicions
- Antibacterial therapy can make visualization of organism very difficult
 Canine 6YO
Color: Brown
Clarity: Opaque
S Color: Lt. tan
S Clarity: Hazy
TP 5.7 mg/dl
TNCC: 30,000/μl
RBC: 230,000/μl

Peritoneum
Direct smear
Modified Wright’s 100x
Modified Wright’s 100x
Modified Wright’s 100x
 6YO MC Dalmatian
Color: Lt. tan
Clarity: Hazy
S Color: Dk. straw
S Clarity: Clear
TP 1.5 mg/dl
TNCC: 9,430/μl
RBC: <30,000/μl

Peritoneum
Diagnostic Peritoneal Lavage
Modified Wright’s 100x
Modified Wright’s 60x
Modified Wright’s 100x
Modified Wright’s 100x
 Cases
Enterocentesis
• Protein- Usually <0.5 g/dl
• Cells- Variable depending on amount of fluid
mixed with gut contents
• Features- Ingesta, assorted debris
- Ingesta, assorted debris
- Mixed population of bacteria
- +/- Protozoa
• Ancillary tests
- Repeat
 12YO F QH
Color: Brown
Clarity: Opaque
S Color: Tan
S Clarity: Hazy
TP 1.0 mg/dl
TNCC: 2,953/μl
RBC: 50,000/μl

Peritoneum
Direct smear
Modified Wright’s 100x
 Cases
Hemorrhage

• Protein- 2.5 - >6.0g/dl, depending on amount of

dilution, duration, etc.
• Cells
- Leukocytes in similar proportion to peripheral blood
- Activated macrophages phagocytizing erythrocytes acutely (6hrs), or
containing hemosiderin and/or hematoidin in chronic situations (72hrs)
- Ancillary tests
- Prussian Blue stain to verify presence of iron-containing hemosiderin
 Cases
Hemorrhage vs. Contamination
ƒ TP 2.5 - >6.0 g/dl depending on
mixture of blood to effusion
ƒ TNCC usually lower the circulation
ƒ WBC morphology looks aged
ƒ RBC much lower than in circulation
ƒ Erythrophagia begins in about 6hrs
ƒ Hemosiderin / hematoidin is
produced in about 72hrs
ƒ TP similar to circulation
ƒ TNCC closer to the periphery
ƒ WBC look similar to circulation
ƒ PLT may be present for around 30
minutes
ƒ RBC / PCV closer to periphery
ƒ No erythrophagia or hemosiderin.
 Canine: 9YO SF
Color: Red
Clarity: Opaque
S Color: Straw
S Clarity: Clear
TP 2.3 mg/dl
TNCC: 2,953/μl
RBC: 4,500,000/μl

Pericardium
Direct smear
Modified Wright’s 100x
Modified Wright’s 100x
Modified Wright’s 100x
Prussian Blue 100x
 Cases
Chylous

• Protein- Lipid interference with refractometer

• Cells
- Acutely, predominantly small, mature lymphocytes
- As duration increases, non-degenerate neutrophils and activate, lipid
laden macrophages appear
• Features
- Will not clear on centrifugation
• Ancillary tests
- Compare fluid to serum triglycerides and cholesterol
 Canine: 10YO MN
Color: White
Clarity: Opaque
S Color: Opal
S Clarity: Opaque
TP: N/A
TNCC: 6,230/μl
RBC: <30,000/μl

Pleura
Direct smear
Modified Wright’s 100x
Modified Wright’s 100x
 Cases
Bilious
• Protein- Variable, but usually >2.5
• Cells
- Moderately to markedly degenerate neutrophils predominate
- Fewer activated macrophages
- Markedly reactive mesothelial cells
• Features
- Phagocytized and free clumps of amorphous blue-grey to yellow pigment
• Ancillary tests
- Prussian Blue to rule out hemosiderin
- Hall’s stain to verify the presence of bile
- Compare fluid bilirubin to serum
 Canine: 7YO MN
Color: Red
Clarity: Semi-op
S Color: Red
S Clarity: Semi-op
TP 3.1 mg/dl
TNCC: 12,953/μl
RBC: <30,000/μl

Peritoneum
Direct smear
Modified Wright’s 20x
Modified Wright’s 100x
 Cases
Neoplastic

• Protein-Variable, but usually >2.5 g/dl

• Cells
- Population of neoplastic cells
- Inflammation confounds interpretation
• Features
- May see lymphoglandular bodies with lymphoma
- May see acinar arrangement with adenocarcinomas
• Ancillary tests
- Locate primary mass
- Immunocytochemistry to identify neoplastic cells
 Feline: 13YO MN
Color: Brown
Clarity: Hazy
S Color: Straw
S Clarity: Hazy
TP 3.1 mg/dl
TNCC: 6,953/μl
RBC: <30,000/μl

Pleura
Direct smear
Modified Wright’s 60x
Modified Wright’s 100x
 Cases
Uroperitoneum
• Protein
- <0.5 g/dl initially
- Increases as duration increases
• Cells
- Low, with aged neutrophils and a few mildly degenerate cells seen
- Absolute number increases with duration
• Features
- May see urinary crystals
• Ancillary tests
- Compare fluid creatinine to serum
- Serum chemistry may show marked hyponatremia, hypochloridemia and
hyperkalemia
 Cases
Feline Infectious Peritonitis
• Protein
- Usually markedly elevated, sometime >8.0 g/dl
- Electrophoresis will show gamma globulins elevated
- A:G <0.8
• Cells
- Mixed inflammatory cells: Non-degenerate neutrophils, activated
macrophages, reactive lymphocytes
• Features
- Coarsely granular protein in the background
• Ancillary tests
- Rivalta test: 5ml distilled water with 1 drop of acetic acid mixed. Add drop
of effusion. A positive test is a gel-like clump that slowly settles
Lily 2002 - 2006