Cytology and Serology Procedures and Interpretation

Cytology: Fine Needle Aspiration and Slide Preparation

Cytology is the microscopic evaluation of cells collected from tissue masses, body fluids, or mucosal surfaces. It is rapid, minimally invasive, and often the first diagnostic step for an unknown mass.

Fine Needle Aspiration (FNA) Technique

FNA collects cells from solid tissue using a small-gauge needle:

• Needle size: 22 to 25 gauge for most cutaneous and subcutaneous masses; 20 to 22 gauge for deeper or firmer tissue.
• Syringe size: 5 to 10 mL if using aspiration; a 3 mL syringe provides adequate vacuum without excess suction.
• Aspiration method: insert the needle into the mass, apply negative pressure by pulling the syringe plunger back 1 to 2 mL, redirect the needle through the mass in several planes while maintaining suction, release the plunger before withdrawing to avoid pulling cells into the syringe barrel.
• Non-aspiration (capillary) method: for vascular or friable tissue, insert the needle without suction and move it through the mass in multiple passes; capillary action collects cells. This reduces blood contamination.
• Expel the sample: detach the needle, fill the syringe with air, reattach the needle, and expel the sample onto a clean slide with a quick push of air.

Slide Preparation Methods

• Squash (compression) prep: place the drop on a slide, gently lower a second slide flat over the drop, allow the blood to spread by capillary action, then pull the slides apart smoothly in parallel or with a slight horizontal slide motion. This spreads cells evenly without crushing them. Use minimal pressure — too much pressure ruptures cells.
• Pull-apart (comb) method: similar to squash but the top slide is lifted straight up — good for very cellular samples.
• Blood smear technique: for fluid samples (effusions, blood-contaminated aspirates), use the same push/wedge method as for blood smears.
• Line smear: for low-cellularity samples, spread most of the drop to one end, concentrating cells in a line for easier evaluation.

Staining and Evaluation

• Diff-Quik is the standard rapid stain for cytology: fixative (5 dips), eosinophilic (3 to 5 dips), basophilic (3 to 5 dips), rinse, dry. Adjust dipping time for sample thickness.
• Wright's-Giemsa provides more nuclear detail for difficult cases; requires longer staining time.
• New methylene blue is used for wet-mount preparations to highlight nuclei and assess cellularity before permanent staining.
• Evaluate under 10x (scan for cellularity and distribution), 40x (identify major cell types), and 100x oil (detail, organisms, nuclear features).

Cytologic Interpretation: Inflammatory vs Non-Inflammatory

The first step in cytologic interpretation is classifying the sample.

Inflammatory Patterns

• Suppurative (neutrophilic): predominantly neutrophils; suggests bacterial infection, sterile inflammation, or acute tissue necrosis. Look for intracellular bacteria to confirm septic versus non-septic.
• Pyogranulomatous: mixture of neutrophils and macrophages; suggests chronic infection (fungal, mycobacterial, foreign body reaction).
• Granulomatous: predominantly macrophages and epithelioid cells; fungal infection, mycobacteria, foreign body.
• Eosinophilic: high proportion of eosinophils; allergic or hypersensitivity reaction, parasitic migration, mast cell tumor, eosinophilic granuloma complex.
• Lymphocytic: predominantly small lymphocytes; chronic antigenic stimulation, early lymphoma.

Non-Inflammatory (Neoplastic) Categories

• Round cell tumors: discrete individual round cells; includes mast cell tumor, lymphoma, histiocytoma, transmissible venereal tumor, plasma cell tumor. Each has distinguishing features (mast cell tumor has purple granules; lymphoma has a monomorphic population of lymphoblasts).
• Epithelial tumors: cells in cohesive sheets or clusters, often with acinar or glandular arrangement; adenomas are uniform, carcinomas show anisocytosis, anisokaryosis, and multiple nucleoli.
• Mesenchymal (spindle cell) tumors: elongated, spindle-shaped cells with tails of cytoplasm; includes sarcomas (fibrosarcoma, liposarcoma, hemangiosarcoma) and benign connective tissue tumors. Cells exfoliate individually and in loose clusters.
• Cysts and normal tissue contaminants: keratin, hair shafts, fat, and normal epithelial cells are not neoplastic and must not be misinterpreted.

Organisms to Identify

Bacteria (cocci, rods, intracellular versus extracellular), fungal hyphae and yeast (Malassezia, Candida, Blastomyces, Histoplasma, Cryptococcus, Coccidioides, Sporothrix), protozoa (Leishmania, Toxoplasma), and parasites may be visible cytologically.

Serology: Principles and Common Veterinary Tests

Serology is the study of serum, specifically the detection of antigens or antibodies. Two fundamentally different approaches exist: antigen detection confirms current infection, while antibody detection (titers) confirms exposure or immune response. Understanding the distinction is essential for interpretation.

Key Distinction: Antigen vs Antibody

• Antigen tests detect a component of the pathogen itself (protein, polysaccharide, cell wall). A positive result means the pathogen is currently present. Example: heartworm antigen test detects a glycoprotein antigen from adult female Dirofilaria immitis.
• Antibody tests (titers) detect the host's immune response to the pathogen. A positive result means the animal has been exposed and mounted an immune response, but does not necessarily mean active infection. Example: Lyme disease antibody test (C6 peptide ELISA) — a positive dog was exposed but may not be clinically infected.
• Vaccine titers (e.g., canine distemper, parvovirus, adenovirus; feline panleukopenia) measure antibody levels to determine whether booster vaccination is needed.

SNAP Tests (Rapid In-House ELISA)

IDEXX SNAP tests are lateral-flow or competitive-flow ELISA devices that deliver results in 8 to 15 minutes. Each test is designed for a specific sample (serum, plasma, or whole blood) and a specific antigen or antibody.

SNAP Test Procedure Pitfalls

• Sample type matters: use the sample specified by the manufacturer. EDTA whole blood works for most SNAP tests; heparinized samples may interfere with some.
• Temperature: refrigerated samples should reach room temperature; cold samples slow antigen-antibody binding.
• Incubation time: follow the exact wait time. Reading too early causes false negatives; reading too late can produce faded spots.
• Flow control: ensure the sample migrates fully across the device; trapped bubbles block flow.
• Positive/negative controls: run controls with each new lot or weekly to verify device integrity.

Titers and Quantitative Serology

A titer is the highest dilution of serum that still produces a detectable reaction. Common titered tests include:

• Leptospira microscopic agglutination test (MAT) — titers of 1:800 or higher in vaccinated dogs and 1:100 or higher in unvaccinated suggest exposure; paired titers 2 to 4 weeks apart showing a 4-fold rise confirm active infection.
• Toxoplasma gondii IgG and IgM — IgM indicates recent infection; IgG indicates past exposure.
• Feline infectious peritonitis (FIP) coronavirus antibody titer — high titers support but do not confirm FIP; diagnosis requires cytology or PCR.
• Babesia canis indirect immunofluorescence (IFA) — used in endemic regions.

ELISA: The Workhorse Immunoassay

Enzyme-linked immunosorbent assay (ELISA) uses an enzyme-linked antibody to produce a color change when antigen-antibody binding occurs. Two formats dominate:

• Direct ELISA: detects antigen in the sample; enzyme-linked antibody binds the antigen directly.
• Indirect ELISA: detects antibody in the sample; antigen is bound to the plate, patient antibody binds the antigen, and a second enzyme-linked anti-species antibody binds the patient antibody.

Color intensity is proportional to the amount of antigen or antibody. Quantitative ELISA uses a standard curve to determine concentration.

Pre-Analytical and Interpretation Pitfalls

• EDTA for hematology, never for chemistry — repeat this mantra: lavender = CBC, red/SST = chemistry, blue = coagulation, green = plasma chemistry, gray = glucose.
• Never freeze whole blood — it lyses cells and invalidates cytology and hematology. Freeze separated serum or plasma only.
• Heartworm antigen test false negatives: prepatent infections (<5 to 6 months post-infection), male-only worm burdens, antigen-antibody complex interference (heat-treat serum to dissociate complexes).
• Antibody vs antigen confusion: an animal that was vaccinated may have antibodies but no active infection; an animal with acute infection may be antibody-negative because the immune response has not yet developed (window period).
• Cross-reactivity: some antibody tests cross-react with related organisms, producing false positives; confirm with a second method when results drive treatment decisions.
• Prozone effect: very high antibody concentrations can overwhelm the assay and produce a false negative; dilute the sample and retest if clinical suspicion is high.