# Cytology of Cutaneous and Subcutaneous Masses: A Diagnostic Approach


## Key Takeaways

- Fine-needle aspiration (FNA) cytology is the primary diagnostic tool for cutaneous and subcutaneous masses in small animals, offering a rapid, minimally invasive, and cost-effective initial assessment that guides further clinical decisions.
- Romanowsky-type stains (e.g., Diff-Quik, Wright-Giemsa) are standard for evaluating cellular morphology, nuclear chromatin, and background material, with air-dried smears prepared immediately after collection to prevent distortion.
- Cytologic diagnosis of neoplasia demonstrates high concordance with histopathology, but false negatives can occur due to sampling error or poor cellularity, necessitating biopsy for persistent or enlarging lesions.
- The diagnostic framework involves classifying samples as inflammatory or neoplastic, then subcategorizing neoplastic cells into round cell, epithelial, or mesenchymal morphologies, each with distinct cytologic features and differential diagnoses.
- Ancillary diagnostic techniques such as immunocytochemistry, flow cytometry, and cytochemistry are crucial for refining diagnoses, particularly for round cell tumors, when morphological assessment is ambiguous.
- Clinical correlation with signalment, lesion history, physical examination findings, and lesion behavior is essential for accurate interpretation and appropriate management planning, including surgical excision, medical therapy, or further diagnostics.

---

Fine-needle aspiration cytology is the first-line diagnostic test for palpable cutaneous and subcutaneous masses in dogs and cats. The procedure is minimally invasive, rapid, and cost-effective, and it can be performed without sedation or analgesia in most patients. Cytology provides immediate results that guide clinical decisions, including whether to pursue surgical excision, medical therapy, or additional diagnostic testing. This article presents a systematic framework for sample collection, slide preparation, and cytologic interpretation, with emphasis on differentiating neoplastic from inflammatory lesions and on recognizing the most common tumor types in small animal practice.

The approach described here serves the practicing veterinarian who performs in-house cytology and must decide when a sample is diagnostic, when it is non-diagnostic, and when ancillary testing such as immunocytochemistry or histopathology is required. Fine-needle aspirates and impression smears of cutaneous and subcutaneous lesions are the most commonly submitted cytology samples from veterinary patients, and diagnostic samples are easily collected without anesthesia or analgesia [MacNeill, institutional publication on cytology of cutaneous and subcutaneous lesions](https://pubmed.ncbi.nlm.nih.gov/21596346/). Cytology can yield immediate results and may prevent the need for additional tests that use more invasive collection methods [MacNeill, institutional publication on cytology of cutaneous and subcutaneous lesions](https://pubmed.ncbi.nlm.nih.gov/21596346/).

Interpretation requires knowledge of the frequency and biological behavior of inflammatory, infectious, and neoplastic lesions, which vary by species, breed, and husbandry conditions [Sharkey et al., review of diagnostic cytology in veterinary medicine](https://pubmed.ncbi.nlm.nih.gov/24554514/). This article covers the technical principles of sample acquisition and preparation, a decision framework for cytologic classification, and the cytologic features that distinguish the major diagnostic categories. Histopathology is excluded from this discussion except where it serves as the reference standard for validating cytologic accuracy.

## At a Glance

| Parameter | Recommendation or Key Fact |
|---|---|
| Sample collection | Fine-needle aspiration with 22- or 25-gauge needle, both gauges yield diagnostic samples |
| Sample types | Fine-needle aspirates, impression smears, swab preparations |
| Staining | Romanowsky-type stains (Wright-Giemsa, Diff-Quik) are standard |
| Diagnostic accuracy | Cytologic diagnosis of neoplasia agrees with histopathology in most cases, false negatives occur |
| Non-diagnostic rate | Approximately 17% of aspirates are non-diagnostic due to poor cellularity |
| Primary classification | Inflammatory, neoplastic, or nondiagnostic, then subclassify |
| Ancillary testing | Immunocytochemistry, cytochemistry, flow cytometry, molecular diagnostics |
| Quality assurance | ASVCP guidelines govern laboratory standards and interpretation |

## Technical Principles of Sample Acquisition

### Needle Selection and Aspiration Technique

Needle gauge influences sample quality but not the overall ability to make a diagnosis. In a randomized comparison of 22- and 25-gauge needles for aspiration of cutaneous, subcutaneous, and intracavitary masses, no significant difference was detected in cellularity between the two gauges, whereas blood contamination, cellular debris, and cellular trauma differed significantly [Arai et al., comparison of 22- vs 25-gauge needles for fine-needle aspiration](https://pubmed.ncbi.nlm.nih.gov/31210389/). The degree of cellular trauma was significantly increased in intracavitary samples, suggesting that tissue consistency and sample site affect cell preservation more than needle gauge does [Arai et al., comparison of 22- vs 25-gauge needles for fine-needle aspiration](https://pubmed.ncbi.nlm.nih.gov/31210389/).

The choice of needle gauge should therefore be based on lesion characteriztics. A 25-gauge needle suits vascular or easily disrupted lesions such as mast cell tumors and lymph nodes. A 22-gauge needle may retrieve more material from fibrous or densely cellular masses. The aspiration technique involves redirecting the needle through the mass multiple times while maintaining negative pressure, then releasing pressure before withdrawing the needle to avoid drawing the sample into the syringe hub.

### Slide Preparation and Staining

Romanowsky-type stains, including Wright-Giemsa and modified Wright stains, are the standard for veterinary cytology [Sharkey et al., review of diagnostic cytology in veterinary medicine](https://pubmed.ncbi.nlm.nih.gov/24554514/). These stains allow evaluation of cytoplasmic granules, nuclear chromatin patterns, and background material such as proteinaceous fluid or infectious organizms. Air-dried smears should be prepared immediately after collection to prevent cell distortion. Impression smears are appropriate for ulcerated or exudative lesions, and swab preparations are useful for draining tracts.

## Diagnostic Accuracy and Limitations

### Concordance with Histopathology

The diagnostic performance of fine-needle aspiration cytology for cutaneous and subcutaneous masses has been evaluated against histopathology as the gold standard. In a retrospective study of 292 palpable masses from dogs and cats, 49 samples were excluded due to poor cellularity, yielding a retrieval rate of 83.2% [Ghisleni et al., correlation between fine-needle aspiration cytology and histopathology](https://pubmed.ncbi.nlm.nih.gov/16511787/). Among the 243 evaluable samples, a cytologic diagnosis of neoplasia was made in 176 cases, with 175 true positives and 1 false positive. Sixty-seven samples were classified as non-neoplastic, including 46 true negatives and 21 false negatives [Ghisleni et al., correlation between fine-needle aspiration cytology and histopathology](https://pubmed.ncbi.nlm.nih.gov/16511787/).

These figures establish two practical points. First, a cytologic diagnosis of neoplasia is highly reliable and rarely represents a false positive. Second, a non-neoplastic cytologic diagnosis does not exclude neoplasia, because sampling error and poor cellularity account for a meaningful proportion of false negatives. A non-diagnostic or inflammatory aspirate from a mass that persists, enlarges, or fails to respond to therapy should prompt biopsy.

### Causes of Non-Diagnostic Samples

Poor cellularity is the most common reason an aspirate cannot be interpreted. Fibrous masses, well-differentiated lipomas, and lesions with extensive necrosis or hemorrhage yield few intact cells. Cystic lesions may yield only fluid. Sampling error occurs when the needle misses the target population, particularly in large or heterogeneous masses. The clinician should aspirate multiple areas of a mass, including the periphery and any solid regions, and should repeat aspiration if the initial smear is paucicellular.

## Cytologic Classification Framework

### Inflammatory Versus Neoplastic

The first interpretive step is to determine whether the sample represents an inflammatory process or a neoplastic population. Inflammatory samples contain a mixed or predominant population of neutrophils, macrophages, lymphocytes, plasma cells, or eosinophils, often with evidence of tissue necrosis or infectious agents. Neoplastic samples contain a relatively monomorphic population of cells that may be round, epithelial, or mesenchymal in morphology. Some neoplasms, particularly those with necrosis or secondary infection, can have a prominent inflammatory component that obscures the underlying neoplastic cells.

### Round Cell, Epithelial, and Mesenchymal Morphology

Once a sample is classified as neoplastic, the cell morphology determines the category. Round cell tumors, including mast cell tumors, lymphoma, plasmacytoma, histiocytic sarcoma, and transmissible venereal tumor, exfoliate readily and contain discrete cells with round nuclei. Epithelial tumors form clusters and sheets with distinct cell borders, variable nuclear to cytoplasmic ratios, and often evidence of acinar or tubular differentiation. Mesenchymal tumors, such as sarcomas, exfoliate poorly and contain spindle-shaped cells with oval nuclei and wispy cytoplasmic borders. The distinction among these categories guides the differential diagnosis and the selection of ancillary tests.

## Quality Assurance and Laboratory Standards

The American Society for Veterinary Clinical Pathology publishes guidelines for quality assurance and laboratory standards in veterinary clinical pathology, including recommendations for sample handling, staining, and interpretation [ASVCP quality assurance and laboratory standards guidelines](https://www.asvcp.org/page/QALS_Guidelines). In-house cytology should follow these standards for stain quality control, slide labeling, and documentation of findings. Samples that are equivocal or that require immunocytochemistry or flow cytometry should be submitted to a reference laboratory with board-certified clinical pathologists, because the application of ancillary diagnostics is complicated by the need to develop and validate species-specific reagents and protocols [Sharkey et al., review of diagnostic cytology in veterinary medicine](https://pubmed.ncbi.nlm.nih.gov/24554514/).

## Pattern-Based Cytologic Classification

The cytologic classification of cutaneous and subcutaneous masses rests on identifying the predominant cell population and its architectural arrangement. Three broad categories emerge: round cell tumors, epithelial tumors, and mesenchymal tumors. Each category carries distinct cytologic features that direct the differential list and guide subsequent diagnostic steps.

Round cell tumors display discrete, individualized cells with round nuclei and a high nuclear-to-cytoplasmic ratio. Cells exfoliate readily and often appear as a monomorphic population. Common round cell tumors in dogs and cats include mast cell tumors, plasmacytomas, lymphomas, histiocytomas, and transmissible venereal tumors. Mast cell tumors warrant particular attention because their cytoplasmic granules may be sparse or absent in poorly granulated variants, and Romanowsky-type stains are essential for granule visualization.

Epithelial tumors exfoliate in clusters, sheets, or acinar arrangements. Cells show distinct cell borders, moderate to abundant cytoplasm, and variable nuclear pleomorphism. Differentiation between benign and malignant epithelial neoplasia relies on criteria such as anisocytosis, anisokaryosis, nuclear molding, prominent nucleoli, and abnormal mitotic figures. Periadnexal tumors, sebaceous adenomas, squamous cell carcinomas, and mammary tumors frequently present with these features.

Mesenchymal tumors yield spindle-shaped or stellate cells with elongated nuclei and poorly defined cytoplasmic borders. Cells often appear in streaming bundles or whorls. Exfoliation is typically sparse compared with round cell tumors, and hemodilution is common. Fibrosarcomas, peripheral nerve sheath tumors, and soft tissue sarcomas fall into this category. The distinction between reactive fibroblasts and neoplastic mesenchymal cells can be challenging, particularly in aspirates with low cellularity.

## Diagnostic Algorithm for Round Cell Tumors

When a round cell population is identified, a systematic algorithm narrows the differential. The algorithm begins with cell morphology and then incorporates cytoplasmic features, nuclear characteriztics, and ancillary testing.

Mast cell tumors show round cells with purple cytoplasmic granules on Wright-Giemsa stain. Eosinophils may accompany the neoplastic population. Poorly granulated mast cell tumors can mimic other round cell tumors, and cytochemical staining for chloroacetate esterase or immunocytochemistry for KIT may be required.

Plasmacytomas display eccentric nuclei, perinuclear clear zones (hofs), and deeply basophilic cytoplasm. Binucleation and multinucleation are common. Amyloid deposition may be present in some cases.

Lymphomas present as a monomorphic population of medium to large lymphocytes with scant cytoplasm. Lymphoblastic morphology, prominent nucleoli, and a high mitotic rate support malignancy. Flow cytometry and immunophenotyping can distinguish B-cell from T-cell origin.

Histiocytomas in dogs show a uniform population of round cells with moderate cytoplasm, round to reniform nuclei, and low mitotic activity. Lymphocyte infiltration is common in regressing lesions. Histiocytic sarcomas display greater pleomorphism and atypia.

Transmissible venereal tumors are characterized by large round cells with distinct borders, abundant vacuolated cytoplasm, and coarsely stippled chromatin. The diagnosis is straightforward in endemic regions.

## Diagnostic Algorithm for Epithelial Tumors

Epithelial cell populations require assessment of arrangement, differentiation, and malignancy criteria. The algorithm prioritizes architectural features first, then cytologic atypia.

Well-differentiated clusters with acinar or tubular formation suggest glandular origin. Squamous differentiation appears as polygonal cells with abundant eosinophilic cytoplasm and occasional keratin pearls. Basaloid cells with high nuclear-to-cytoplasmic ratios and peripheral palisading suggest adnexal tumors.

Malignancy criteria in epithelial tumors include marked anisocytosis, anisokaryosis, nuclear crowding, prominent or multiple nucleoli, and atypical mitotic figures. The presence of these features supports a carcinoma diagnosis. However, well-differentiated carcinomas may show minimal atypia, and inflammatory atypia can mimic neoplasia. Correlation with clinical behavior and histopathology remains necessary for definitive grading.

## Diagnostic Algorithm for Mesenchymal Tumors

Mesenchymal aspirates require careful evaluation of cellularity, cell shape, and matrix production. The algorithm begins with cellularity assessment because low cellularity limits interpretation.

Spindle cells with elongated nuclei and wispy cytoplasm suggest fibrosarcoma or peripheral nerve sheath tumor. Myxoid background material supports myxosarcoma or myxoid liposarcoma. Liposarcomas show variably vacuolated cells with scalloped borders and may contain lipid droplets. Hemangiopericytomas and perivascular wall tumors display whorling patterns and perivascular arrangement.

Malignancy criteria for mesenchymal tumors include marked nuclear atypia, high nuclear-to-cytoplasmic ratio, and pleomorphism. Mitotic figures are uncommon in aspirates, so their absence does not exclude malignancy. The distinction between benign and malignant mesenchymal proliferations is often impossible on cytology alone, and histopathology is recommended for definitive diagnosis.

## Ancillary Diagnostic Techniques

Ancillary testing can refine cytologic diagnoses when morphology is ambiguous. Immunocytochemistry on direct smears or cytospin preparations can identify cell lineage using antibodies against CD3, CD20, CD79a, cytokeratin, vimentin, and KIT. Flow cytometry is particularly useful for lymphoma phenotyping and can detect aberrant antigen expression. Cytochemistry, such as chloroacetate esterase for mast cells, provides additional confirmation.

Molecular diagnostics, including PCR for antigen receptor rearrangements and mutation analysis, may be applied to cytologic samples. These techniques require adequate cellularity and appropriate sample handling. The [ASVCP quality assurance guidelines](https://www.asvcp.org/page/QALS_Guidelines) emphasize the importance of standardized protocols and validation for ancillary testing in veterinary cytology.

## Decision Points and Clinical Correlation

Cytologic findings must be integrated with signalment, lesion history, and physical examination. Breed predispositions influence the differential list. For example, mast cell tumors are common in Boxers and Pugs, while histiocytomas occur predominantly in young dogs. Cats with cutaneous masses require consideration of vaccine-associated sarcomas and mammary tumors.

Lesion location and growth rate provide additional context. Rapidly growing masses with ulceration or fixation to underlying tissues raise concern for malignancy. Multiple cutaneous nodules suggest metastatic disease, systemic neoplasia, or an inflammatory process such as panniculitis.

The decision to proceed with surgical excision, biopsy, or medical management depends on the cytologic diagnosis and clinical staging. A cytologic diagnosis of mast cell tumor warrants staging with lymph node aspiration and abdominal ultrasound. A diagnosis of lipoma may allow conservative management, whereas a liposarcoma requires wide surgical excision.

## Documentation and Reporting

Cytologic reports should include sample quality, cellularity, cell population description, and a cytologic interpretation. The interpretation should state whether the findings are consistent with inflammation, neoplasia, or a specific tumor type. When a definitive diagnosis is not possible, the report should list differential diagnoses and recommend additional testing.

Standardized terminology improves communication between clinicians and pathologists. The [MSD Veterinary Manual](https://www.msdvetmanual.com/) and [AVMA practice resources](https://www.avma.org/resources-tools) provide frameworks for consistent reporting and clinical decision support. Reports should note the limitations of cytology, particularly when sample cellularity is low or when benign and malignant mesenchymal lesions cannot be distinguished.

Table 1 summarizes the cytologic features that guide classification and the recommended next steps for each category.

| Cell Population | Key Cytologic Features | Common Diagnoses | Recommended Next Step |
| --- | --- | --- | --- |
| Round cells | Discrete cells, round nuclei, high N:C ratio | Mast cell tumor, lymphoma, plasmacytoma, histiocytoma | Immunocytochemistry, flow cytometry, staging |
| Epithelial cells | Clusters, sheets, acini, distinct borders | Carcinoma, adenoma, squamous cell carcinoma | Histopathology for grading, surgical excision |
| Mesenchymal cells | Spindle cells, streaming, low cellularity | Fibrosarcoma, liposarcoma, peripheral nerve sheath tumor | Histopathology for definitive diagnosis |
| Mixed population | Inflammatory cells with reactive epithelium | Abscess, granuloma, inflamed neoplasia | Culture, biopsy if non-responsive |

Species differences affect the frequency and behavior of specific tumors. Canine cutaneous histiocytomas are typically benign and self-limiting, while feline histiocytic disorders carry a poorer prognosis. The [cytologic review of canine and feline cutaneous lesions](https://pubmed.ncbi.nlm.nih.gov/21596346/) highlights these species-specific differences and their impact on diagnostic interpretation.

When cytology yields a non-diagnostic sample, repeat aspiration with a larger needle or ultrasound guidance may improve cellularity. Studies comparing needle gauges found that 22-gauge and 25-gauge needles produce comparable diagnostic quality, although smaller needles cause less blood contamination and cellular trauma. The [comparative study of needle gauges](https://pubmed.ncbi.nlm.nih.gov/31210389/) supports using 25-gauge needles for vascular or fragile lesions and 22-gauge needles when greater cellularity is needed.

In endemic regions, infectious causes must be considered. Coccidioidomycosis can present as subcutaneous nodules in dogs and cats, and cytology frequently identifies the spherules. The [retrospective analysis of coccidioidomycosis cases](https://pubmed.ncbi.nlm.nih.gov/27397725/) found that cytology was the most common diagnostic method for cutaneous lesions. Similarly, Dirofilaria repens infestation produces subcutaneous nodules containing nematode cross-sections and microfilariae, as described in [canine nodular dirofilariosis](https://pubmed.ncbi.nlm.nih.gov/23413808/). These infectious etiologies underscore the importance of considering regional pathogens in the differential diagnosis of cutaneous masses.

## Recognized Complications and Early Detection

Fine-needle aspiration is a low-morbidity procedure, but complications do occur. Hemorrhage is the most common, particularly with vascular masses such as haemangiosarcoma or highly cellular round cell tumors. Most hemorrhage is self-limiting and confined to the subcutaneous space. Early detection relies on applying firm manual pressure for two to three minutes after sampling, then re-palpating the site before the patient is discharged. Expanding swelling, pain on palpation, or a drop in mucous membrane color warrants immediate reassessment.

Needle tract seeding is a recognized but uncommon complication when aspirating malignant neoplasms. The risk is highest for mast cell tumors and carcinomas. Detection is delayed, often by weeks to months, and presents as a new nodule along the previous needle path. The practical consequence is that the aspiration site should be recorded in the medical record, and surgical excision planning should include the needle tract when malignancy is confirmed.

Pneumothorax is possible when aspirating masses over the thoracic wall, particularly in thin patients or with deep subcutaneous lesions. Tachypnoea, dull lung sounds, or progressive respiratory effort within hours of sampling should prompt thoracic imaging. The risk is reduced by using a 25-gauge needle, limiting the number of passes, and avoiding perpendicular penetration of the intercostal space [Fine-needle aspiration of cutaneous, subcutaneous, and intracavitary masses in](https://pubmed.ncbi.nlm.nih.gov/31210389/).

Infection is rare with proper skin preparation, but immunosuppressed patients or those with contaminated lesions are at higher risk. Cellulitis, heat, or purulent discharge developing within 48 hours warrants culture and systemic antimicrobial therapy.

## Common Interpretation Errors and Corrective Action

The most frequent error is over-interpreting a poorly cellular sample. A diagnosis of neoplasia should never be rendered from fewer than three to five well-preserved cell groups unless the cells are unequivocally diagnostic, such as mast cells with visible granules. The corrective action is to repeat the aspirate or use an alternative collection method instead of force a conclusion from inadequate material.

Misclassifying inflammation as neoplasia occurs when reactive mesenchymal cells or epithelioid macrophages are mistaken for tumor cells. Reactive fibroblasts can be large, pleomorphic, and binucleate. The discriminating feature is the mixed population: inflammation shows a spectrum of cell types, whereas neoplasia tends toward a monomorphic population. When in doubt, the sample should be reported as inflammatory with reactive atypia, and histopathology recommended.

The reverse error, calling a neoplasm inflammatory, happens most often with mast cell tumors that have undergone degranulation. The granules may be absent or sparse, leaving only sheets of round cells that resemble macrophages or lymphocytes. A Diff-Quik stain with prolonged staining time, or a second slide stained with toluidine blue, can reveal metachromatic granules. This distinction matters because mast cell tumor cytology is highly reliable when granules are present, and the treatment pathway diverges sharply from inflammatory disease [Cytology of canine and feline cutaneous and subcutaneous lesions](https://pubmed.ncbi.nlm.nih.gov/21596346/).

A third error is failing to correlate cytology with clinical behavior. A cytologically benign lipoma that is rapidly growing, painful, or adherent to underlying tissue should not be accepted at face value. Liposarcoma can be cytologically similar to lipoma in well-differentiated forms. Clinical discordance is an indication for biopsy, not for reassurance.

## Troubleshooting Table

| Observation | Likely Cause | Discriminating Check |
|---|---|---|
| Acellular smear, blood only | Missed lesion, cystic fluid, or vascular sampling | Re-palpate, aspirate the center and the capsule separately |
| Thick, clumped smear | Excessive aspiration pressure or smearing force | Prepare a second slide with gentler technique, assess for crush artefact |
| Cells all ruptured | High vacuum, small needle lumen, or delayed smearing | Use 22-gauge needle for fragile round cells, smear immediately [Fine-needle aspiration of cutaneous, subcutaneous, and intracavitary masses in](https://pubmed.ncbi.nlm.nih.gov/31210389/) |
| Granules absent in suspected mast cell tumor | Degranulation during sampling or staining | Repeat with toluidine blue, check for eosinophils as a clue |
| Mixed inflammatory and neoplastic cells | Concurrent inflammation or tumor necrosis | Report both components, recommend biopsy for definitive classification |
| Abundant extracellular matrix | Mesenchymal tumor, myxoma, or myxosarcoma | Assess nuclear atypia, matrix alone does not confirm malignancy |

## Limitations of Current Evidence

The published evidence on cytologic accuracy in cutaneous masses is moderate in size and largely retrospective. The largest comparative study reported an 83.2% retrieval rate and a cytologic diagnosis of neoplasia in 176 of 243 evaluable samples, with one false positive and 21 false negatives against histopathology [Correlation between fine-needle aspiration cytology and histopathology in the](https://pubmed.ncbi.nlm.nih.gov/16511787/). These figures are useful but derive from a single institutional population, and accuracy varies with tumor type, sample quality, and the experience of the interpreter.

Expert opinion still differs on several points. The value of routine ancillary testing, such as immunocytochemistry or flow cytometry, is accepted for round cell tumors but the threshold for ordering these tests varies widely between institutions [All lesions great and small, part 1: diagnostic cytology](https://pubmed.ncbi.nlm.nih.gov/24554514/). Some clinicians advocate cytology for every palpable mass before any other test, while others reserve it for lesions with atypical features. Neither position is wrong, but the choice should reflect the clinical question and the likelihood that cytology will change management.

The evidence base for needle gauge selection is similarly limited. One prospective study found no significant difference in diagnostic ability between 22- and 25-gauge needles, despite differences in blood contamination and cellular trauma [Fine-needle aspiration of cutaneous, subcutaneous, and intracavitary masses in](https://pubmed.ncbi.nlm.nih.gov/31210389/). This supports practitioner preference, but the study was small and included intracavitary masses, which may not reflect subcutaneous lesions alone.

## Referral, Consultation, and Reporting Thresholds

Referral for histopathology is indicated when cytology is non-diagnostic, when cytologic and clinical findings conflict, or when the cytologic diagnosis is malignant and surgical planning requires margin assessment. Specialist consultation with a clinical pathologist is appropriate for challenging round cell tumors, for samples with mixed populations, and before ancillary testing is ordered, because sample preparation requirements differ between immunocytochemistry and flow cytometry [All lesions great and small, part 1: diagnostic cytology](https://pubmed.ncbi.nlm.nih.gov/24554514/).

Laboratory involvement is warranted when infectious agents are suspected but not identified, particularly for fungal disease. Coccidioidomycosis is frequently diagnosed by cytology in endemic regions, but culture or serology may be needed for speciation and treatment planning [Retrospective analysis of cutaneous lesions in 23 canine and](https://pubmed.ncbi.nlm.nih.gov/27397725/). Parasitic nodules, such as those caused by Dirofilaria repens, may show nematode fragments on cytology but require molecular confirmation for species identification [Nodular lesions due to infestation by Dirofilaria repens in](https://pubmed.ncbi.nlm.nih.gov/23413808/).

Regulatory reporting is rarely triggered by cytology alone. However, lesions consistent with notifiable diseases, including vesicular or ulcerative lesions in livestock or wildlife, should be reported according to regional animal health requirements. The World Organization for Animal Health maintains current lists of notifiable diseases, and practitioners should consult these standards when clinical signs suggest a reportable condition [WOAH terrestrial animal health standards](https://www.woah.org/en/what-we-do/standards/codes-and-manuals/terrestrial-code-online-access/).

## Frequently Asked Questions

### How should I proceed when a mass yields a non-diagnostic sample but remains clinically suspicious?

Repeat the aspiration, ideally with a different needle gauge and sampling angle. The [comparison of 22- versus 25-gauge needles](https://pubmed.ncbi.nlm.nih.gov/31210389/) found no significant difference in diagnostic yield, so switching gauge is less important than ensuring adequate sampling technique. If a second attempt is acellular, consider ultrasound guidance for deeper lesions or a different collection method such as a wedge biopsy or punch biopsy for histopathology. A non-diagnostic cytology result does not exclude neoplasia. In one series, 21 of 67 cytologically non-neoplastic samples were false negatives on histopathology, so persistent clinical suspicion warrants tissue biopsy even when cytology is unrewarding [correlation between fine-needle aspiration cytology and histopathology](https://pubmed.ncbi.nlm.nih.gov/16511787/).

### What cytologic findings should prompt screening for systemic fungal disease in endemic regions?

In endemic areas such as the southwestern United States, coccidioidomycosis can present as subcutaneous nodules with or without systemic illness. Cytology identified the organizm in most confirmed cases in one retrospective series, and 75% of dogs and 54.5% of cats with cutaneous lesions had concurrent systemic signs [retrospective analysis of cutaneous coccidioidomycosis in dogs and cats](https://pubmed.ncbi.nlm.nih.gov/27397725/). Look for spherules with endospores against a pyogranulomatous background. When cytology reveals these structures, obtain thoracic radiographs and serologic testing to stage dissemination. Localized lesions without systemic illness occur and may represent primary cutaneous inoculation, but the presence of spherules on cytology still warrants systemic evaluation before surgical planning.

### How do I handle a subcutaneous nodule that contains nematode larvae or cross-sections?

Nematode fragments in a cytologic preparation from a subcutaneous nodule should raise suspicion for Dirofilaria repens infestation, particularly in dogs with travel history to endemic regions of Europe or the Mediterranean basin. In one case series, cytology identified microfilariae in nodules from 12 of 16 affected dogs, and concurrent infection with Dirofilaria immitis was present in some cases [nodular lesions due to Dirofilaria repens infestation in dogs](https://pubmed.ncbi.nlm.nih.gov/23413808/). Perform a modified Knott test to screen for circulating microfilariae and distinguish species. Adult worms may be coiled within the nodule, so excisional biopsy is both diagnostic and therapeutic. Discuss zoonotic implications with the owner, as D. repens can cause human dirofilariosis.

### What is the minimum equipment needed to obtain diagnostic cytology in a general practice setting?

A 22- or 25-gauge needle, a 3 to 6 mL syringe, glass slides, and a Romanowsky-type stain such as Diff-Quik are sufficient for most cutaneous and subcutaneous masses. The [ASVCP quality assurance guidelines](https://www.asvcp.org/page/QALS_Guidelines) emphasize that sample quality depends more on technique than on specialized equipment. If a syringe is unavailable, needle-only aspiration without suction often yields adequate cellularity for soft, vascular masses. For cystic or fluid-filled lesions, collect fluid into an EDTA tube for cell counts and protein measurement. If in-house staining is unreliable, submit air-dried unstained slides to a reference laboratory, as delayed staining of properly dried slides does not compromise diagnostic quality.

### How should cytology findings be documented in the medical record?

Record the mass location, size, gross appearance, and the collection method used. Describe smear cellularity, background material such as blood or proteinaceous fluid, and the predominant cell population with a stated level of confidence. Include a differential diagnosis list ranked by cytologic support. Note whether the sample was adequate for interpretation and whether ancillary testing such as immunocytochemistry or culture was recommended. The [MSD Veterinary Manual](https://www.msdvetmanual.com/) advises that cytology reports should communicate both the cytologic interpretation and its limitations. If samples are submitted to a reference laboratory, retain the in-house preparation for comparison and document the submission date and laboratory accession number.

### How do I explain cytologic uncertainty to an owner without undermining confidence in the diagnostic plan?

Frame cytology as a screening test that guides the next diagnostic step, not as a final diagnosis. Explain that cytology examines individual cells, whereas histopathology examines tissue architecture, and that some tumors require the latter for definitive classification. The [correlation study between cytology and histopathology](https://pubmed.ncbi.nlm.nih.gov/16511787/) reported an 83.2% retrieval rate and occasional false negatives, so owners should understand that a benign cytologic result does not guarantee benign behavior. Use concrete language: "The cells look like a lipoma, but we cannot rule out a low-grade sarcoma without biopsy." Offer a clear plan, including timing for recheck and the specific circumstances under which biopsy would be recommended.

## Related Clinical & Scientific Guides

* [Peripheral Blood Smear Evaluation: A Step-by-Step Guide](/knowledge/veterinary-medicine/clinical-pathology/peripheral-blood-smear-evaluation-guide)
* [Reticulocyte Counts in Veterinary Medicine: Clinical Utility and Interpretation](/knowledge/veterinary-medicine/clinical-pathology/reticulocyte-counts-veterinary-medicine)
* [Cerebrospinal Fluid Analysis in Veterinary Neurology: Collection and Interpretation](/knowledge/veterinary-medicine/clinical-pathology/cerebrospinal-fluid-analysis-veterinary)


## References and Further Reading

- [Cytology of canine and feline cutaneous and subcutaneous lesions and lymph nodes.](https://pubmed.ncbi.nlm.nih.gov/21596346/). 2011.
- [All lesions great and small, part 1: diagnostic cytology in veterinary medicine.](https://pubmed.ncbi.nlm.nih.gov/24554514/). 2014.
- [Correlation between fine-needle aspiration cytology and histopathology in the evaluation of cutaneous and subcutaneous masses from dogs and cats.](https://pubmed.ncbi.nlm.nih.gov/16511787/). 2006.
- [Retrospective analysis of cutaneous lesions in 23 canine and 17 feline cases of coccidioidomycosis seen in Arizona, USA (2009-2015).](https://pubmed.ncbi.nlm.nih.gov/27397725/). 2016.
- [Fine-needle aspiration of cutaneous, subcutaneous, and intracavitary masses in dogs and cats using 22- vs 25-gauge needles.](https://pubmed.ncbi.nlm.nih.gov/31210389/). 2019.
- [Nodular lesions due to infestation by Dirofilaria repens in dogs from Italy.](https://pubmed.ncbi.nlm.nih.gov/23413808/). 2013.
- [American Society for Veterinary Clinical Pathology Guidelines](https://www.asvcp.org/page/QALS_Guidelines). American Society for Veterinary Clinical Pathology.
- [MSD Veterinary Manual, Professional Edition](https://www.msdvetmanual.com/). MSD Veterinary Manual.
- [American Veterinary Medical Association Practice Resources](https://www.avma.org/resources-tools). American Veterinary Medical Association.

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> This article is educational professional reference material for veterinary audiences. It is not a substitute for veterinary diagnosis, individual clinical judgment, current product labeling, or applicable regulatory requirements.