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Fundamentals of Clinical Cytology:
• Purpose and Scope
◦ Serves as a clinical tool to investigate disease processes, primarily in small animals but applicable to all species.
◦ Should be viewed as a guide rather than a final word; definitive diagnosis or assessment of complex therapy often requires histopathology to examine tissue architecture.
• Sample Quality and Staining
◦ Full interpretation requires high-quality samples; practitioners are encouraged to stain one slide in-house to monitor quality.
◦ Stains: Traditional stains include modified Wright and May-Grunwald Giemsa, though rapid Romanowsky stains are now common in practice.
◦ Special Cases: Formalin-fixed preparations require H&E or Papanicolaou stains. Stains deteriorate with use and must be renewed regularly.
Collection Techniques
◦ Fine-needle Aspiration (FNA): Uses 21- to 25-gauge needles and 3- to 10-mL syringes.
▪ Suction Technique: Needle is redirected while applying suction; suction must be released before withdrawal to avoid cell rupture.
▪ Non-aspiration (Capillary) Technique: Uses a needle without a syringe; cells enter via capillary action, which is superior for fragile lymphoid cells and splenic lesions.
◦ Impression Smears: Used for ulcerated surfaces or excised biopsy samples. Cut surfaces should be blotted to remove blood/serum before being applied to the slide.
◦ Body Fluids: Best concentrated via cytospin or centrifugation. Adding a few drops of formalin preserves cells for transit and prevents bacterial overgrowth.
Interpretation of Inflammation
• Primary Cell Types
◦ Neutrophils: First to arrive; large numbers indicate acute inflammation often caused by infection or foreign bodies. Pathogenic bacteria should be found within the cytoplasm to confirm they are not contaminants.
◦ Macrophages: Arrive within 2–3 hours; they phagocytize larger structures like fungi and debris. Activated macrophages appear vacuolated, while epithelioid cells (uniform cytoplasm) can mimic epithelial cells but do not form clusters.
◦ Eosinophils: Associated with allergies, parasites, and specific conditions like eosinophilic granuloma in cats or mast cell tumors in dogs.
◦ Lymphocytes/Plasma Cells: Indicate a chronic response, arriving days after acute inflammation.
◦ Multinucleated Cells: Large cells developed from macrophages, typically seen late in granulomatous reactions.
• Reactive Fibroblasts
◦ Proliferate during tissue repair (approx. 2 days post-injury); they are elongated with "wispy" cytoplasm.
◦ Cytologically, they are difficult to distinguish from low-grade spindle cell tumors.
Interpretation of Neoplasia:
• Basic Tissue Types
◦ Epithelial: Round or polygonal cells that exfoliate in clusters or sheets with sharp outlines.
◦ Mesenchymal (Spindle Cells): Tightly adherent cells that exfoliate in low numbers; they often appear "wispy" and round up once removed from tissue.
◦ Round Cells: Have no adherence and exfoliate in large numbers as individual cells. Includes mast cells, lymphocytes, histiocytes, and plasma cells.
• Criteria for Malignancy
◦ Nuclear features are the primary indicators: increased nuclear size, high N:C ratio, coarse/clumped chromatin, nuclear moulding, and large/multiple/irregular nucleoli.
◦ General variation: Increased mitosis with abnormal figures and marked pleomorphism (variation in size and shape).
• Important Exceptions
◦ Thyroid Carcinomas: Often have uniform, well-differentiated cells; malignancy is often judged by mass size in dogs.
◦ Lymphoma: Characterized by a uniform population of large immature cells (>50%) rather than morphological variation.
◦ Mammary Tumors: Cellular morphology is an unreliable guide; behavior is best assessed via local tissue invasion (histology).
Analogy for Understanding Malignancy Criteria: Think of a healthy tissue as a well-organized choir where everyone is the same size, wearing the same uniform, and following the same conductor. In a malignant tumor, it is as if the choir members have gone rogue: they are all different sizes (pleomorphism), some are wearing three hats (multinucleation), they are shouting at different volumes (clumped chromatin), and they are stepping out of line to crowd their neighbors (nuclear moulding).
Common Cytological Findings and Artifacts
• Mature Fat Cells: Diagnostic for lipoma if sampled from a nodular mass; they appear as clear-staining, "balloon-like" cells.
• Keratin: Can be a contaminant from skin or sampled from cutaneous cysts (always benign).
• Blood: A common artifact of FNA; the presence of platelets suggests the blood came directly from the vascular system during sampling.
• Cytoplasmic Granules:
◦ Mast Cells: Dark blue/purple metachromatic granules.
◦ Melanin: Large black granules in melanocytes/melanomas or macrophages.
◦ Bile: Appears as dark ribbons (canalicular plugs) or dark-staining bodies in hepatocytes.
Site-Specific Cytology
I. Lymph Nodes
• Normal vs. Hyperplastic: Features a mixed population of cells, predominantly small lymphocytes with variable medium/large lymphoid cells and plasma cells.
• Neoplasia (Lymphoma):
◦ Indicated by a uniform population of cells.
◦ Nuclear Size: Neoplastic cells have nuclei at least 1.5 times the diameter of RBCs.
◦ Diagnostic Threshold: Most cytologists diagnose lymphoma when immature cells are >50% of the population.
◦ Grading: Histopathologic confirmation is essential for full grading and therapy planning.
• Submandibular Node Challenges:
◦ Frequently undergo atypical hyperplasia due to antigenic stimuli from the buccal/nasal areas.
◦ Prone to false negatives/positives; often yield only salivary cells (large foamy cells) due to sampling error.
II. Body Cavity Fluids
• Analysis Requirements: Must include total protein (refractometer), total cell count, and a differential.
• Classification:
◦ Transudate: Protein < 2.5 g/dL; Cell Count < 1,500 cells/mL.
◦ Modified Transudate: Protein 2.5–7.5 g/dL; Cell Count 1,000–7,000 cells/mL.
◦ Exudate: Protein > 3.0 g/dL; Cell Count > 7,000 cells/mL.
• Mesothelial Cells:
◦ Proliferate and shed when fluid accumulates; can appear malignant (large, multinucleated, clusters).
◦ Distinguishing Feature: Often have a corona (fuzzy outline) around the cytoplasmic envelope.
◦ Malignancy: It is impossible to differentiate neoplastic mesotheliomas from reactive mesothelial cells via cytology alone.
III. Respiratory (Tracheal or BAL)
• Healthy Animals: Macrophages are the dominant cell type.
• Inflammatory Indicators:
◦ Neutrophils: Indicate inflammation; intracellular bacteria confirm significant infection.
◦ Eosinophils: Indicate allergic respiratory disease, lungworms, or heartworms.
• Contamination: Nucleated/nonnucleated squamous cells with bacteria (specifically the large, ladder-like Simonsiella) confirm contamination from the pharynx.
IV. Synovial Fluid
• Characteristics: Normal fluid is viscous and sticky.
• Artifactual vs. True Hemarthrosis:
◦ Artifact: Blood appears at the end of sampling; clear fluid initially.
◦ True: Uniformly bloody; RBCs found inside macrophage cytoplasm.
• Cytologic Features:
◦ Windrowing: Cells align in rows during smearing due to normal or increased viscosity.
◦ Cell Counts: Normal counts are low (~2 cells/400X field).
◦ Disease Patterns: Degenerative joint disease shows low cell numbers (mostly macrophages); septic or autoimmune arthritis shows very high cell numbers (mostly neutrophils).
V. Nasal Cavity
• Cell Population: Similar to BAL; includes respiratory epithelial cells and exudate.
• Eosinophils: Less specific than in the lower airway; may indicate allergens, parasites, fungi, bacteria, or neoplasia.
• Limitations: Neoplasia and fungal infections are often missed unless the lesion erodes the epithelium or plaques are sampled directly.
VI. Vaginal Cytology
• Epithelial Cell Progression: Parabasal → Small Intermediate → Large Intermediate → Superficial (pyknotic or absent nuclei).
• Cycle Stages:
◦ Proestrus: All cell types present; neutrophils and RBCs common; neutrophils decrease as it progresses.
◦ Estrus: >90% superficial cells; large numbers of bacteria; no neutrophils.
◦ Diestrus: >80% parabasal and intermediate cells; neutrophils return.
◦ Anestrus: Predominantly parabasal and intermediate cells; rare neutrophils/bacteria.
VII. Cerebrospinal Fluid (CSF)
• Handling: Cells degenerate rapidly due to low albumin; samples should be examined within 1 hour.
• Pleocytosis: An increase in nucleated cells.
• Interpretation: Neutrophils/macrophages should be searched for organisms; however, absence of pleocytosis does not exclude infection.
VIII. Urine Cytology
• Preparation: Dried smears are better than wet preparations for identifying nucleated cells.
• Preservation: Boric acid or a few drops of formalin (requires H&E stain) can be used for delayed examination.
• Findings:
◦ Neoplastic Cells: Almost always epithelial; rounded polygonal cells, often clumped with high pleomorphism.
◦ Inflammation: Mostly neutrophils (cystitis); bacteria must be sought within the cytoplasm.
◦ Interstitial Cystitis: Suggested by persistent hematuria without cytologic evidence of inflammation or neoplasia.
IX. Liver Cytology
• Limitations: Blood contamination is common; many diseases require architectural assessment (histology).
• Hepatocyte Changes:
◦ Lipidosis: Numerous small or large discrete vacuoles.
◦ Steroid Induction: Enlarged cells with pale, granular-staining cytoplasm (glycogen) but no vacuoles.
◦ Bile Stasis: Black ribbons (canalicular plugs) or larger dark-staining bodies in the cytoplasm.
• Nodular Lesions: Difficult to differentiate benign hyperplasia or adenoma from normal hepatocytes or well-differentiated carcinoma.
• Round Cell Tumors: Cytology is highly useful for diagnosing lymphoma (medium/large lymphoid cells) and mast cell tumors (metachromatic granules).
X. Kidney Cytology
• Normal: Predominantly uniform renal tubular cells; cats often have normal lipid droplets in the cytoplasm.
• Neoplasia: Renal lymphoma is the most common and easily diagnosed due to wide distribution.
• Inflammation: Chronic lesions often yield low cell counts due to fibrous tissue; cytology is generally not useful for these.
XI. Mammary Cytology
• Utility: Best for differentiating inflammatory vs. neoplastic nodular lesions.
• Prognosis: Cellular morphology is a poor guide for tumor behavior; local tissue and vessel invasion (assessed via histology) are the primary prognostic indicators.
• Species Note: In cats, malignant cells are often uniform; tumor size is a more useful prognostic indicator than morphology.
Analogy for Understanding Mesothelial Cells: Think of mesothelial cells like reactive neighbors during a storm (fluid accumulation). Normally, they stay quiet and out of sight. When the storm hits, they may puff out their chests, gather in groups, and look intimidating (malignant). However, their "fuzzy" jackets (the corona) often give them away as just being reactive to the weather, rather than being actual "intruders" (neoplastic cells).
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