Cytologic Differentiation of Large Cell and Small Cell Lymphoma in Dogs

Lymphoma remains one of the most common hematopoietic malignancies in dogs, with cytologic evaluation serving as a critical first-line diagnostic tool. While histopathology with immunophenotyping provides definitive classification, cytology offers rapid, minimally invasive assessment that can guide initial treatment decisions and prognostic discussions. Understanding the cytomorphologic features that distinguish large cell from small cell lymphoma is essential for accurate case management and prognostic counseling.

Classification Systems Overview

Cytologic classification of canine lymphoma is based primarily on cell size and morphology. The Updated Kiel classification (later adopted by WHO) uses morphologic criteria to categorize lymphomas, with cell size being a fundamental distinguishing feature. Large cell lymphomas generally correspond to higher-grade behavior, while small cell lymphomas typically exhibit more indolent clinical courses. The Camus cytological grading system, validated specifically for cytologic samples, incorporates cell size as a primary criterion for classification of canine lymphomas on fine-needle aspirate preparations.

Feature	Large cell (high-grade)	Small cell (low-grade)
Nuclear characteristics
Nuclear size	>1.5-2× neutrophil diameter	1-1.5× neutrophil diameter
N:C ratio	>0.7 (high ratio, minimal cytoplasm)	<0.6 (lower ratio, more abundant cytoplasm)
Nuclear contours	Variable, irregular membranes, clefting, convolutions	Round to oval, regular membranes
Chromatin pattern	Fine to coarse, dispersed or clumped	Condensed, uniformly mature appearance
Nucleoli	Prominent (1-3 large nucleoli)	Absent or inconspicuous
Cellular features
Anisocytosis	Marked variation in cell and nuclear size	Minimal variation, uniform appearance
Mitotic index	High (frequent mitotic figures)	Low (rare to absent mitoses)
Cell integrity	Fragile cells, numerous naked nuclei	Well-preserved cytoplasm, intact cells
Morphology	Immature, lymphoblastic appearance	Mature lymphocyte appearance
Sample characteristics
Cellularity	High cellularity, densely packed	Variable cellularity
Population	Monomorphic (loss of heterogeneity)	Monotonous, very uniform
Background	Tingible body macrophages, significant debris	Minimal debris and necrosis
Common subtypes
Examples	Diffuse large B-cell lymphoma (DLBCL) Lymphoblastic lymphoma Burkitt-like lymphoma	Marginal zone lymphoma Follicular lymphoma Small lymphocytic lymphoma/CLL T-zone lymphoma

Diagnostic Challenges and Pitfalls

Reactive Hyperplasia vs. Low-Grade Lymphoma

Low-grade lymphomas can be particularly challenging to distinguish from reactive lymphoid hyperplasia:

• Heterogeneity: Reactive nodes show mixed population of small lymphocytes, plasma cells, and macrophages

• Architecture: Reactive nodes may retain some follicular architecture

• Clinical correlation: Essential to consider signalment, clinical signs, and imaging findings

• Flow cytometry: Can help demonstrate monoclonality in equivocal cases

Intermediate Cases

Some lymphomas display mixed features or fall into intermediate grades:

• Consider additional diagnostics: Histopathology, immunophenotyping, and PARR (PCR for Antigen Receptor Rearrangement)

• Document uncertainty: Clear communication about diagnostic confidence is crucial

Sample Quality Issues

• Hemodilution: Can make assessment difficult; use feathered edge for evaluation

• Crush artifact: May artificially increase nuclear irregularity

• Air-drying artifact: Can alter chromatin appearance; evaluate well-prepared areas

Clinical Implications

Prognostic Significance

• High-grade lymphomas: Typically more aggressive but may respond better to chemotherapy; median survival times often 6-12 months with treatment

• Low-grade lymphomas: Generally slower progression; some subtypes (T-zone) may have prolonged survival (2-4+ years) even with minimal treatment

Treatment Considerations

• High-grade: Multi-agent chemotherapy protocols (CHOP-based) are standard of care

• Low-grade: Treatment approaches vary; some indolent forms may warrant "watch and wait" approach or single-agent therapy

Prognostic Factors Beyond Grade

• Immunophenotype: B-cell vs. T-cell origin significantly impacts prognosis

• Anatomic location: Multicentric vs. extranodal (GI, cutaneous, etc.)

• Stage: Extent of disease involvement

• Substage: Presence or absence of clinical signs

Practical Approach to Cytologic Evaluation

1. Systematic assessment:

• Scan at low power for cellularity and architecture

• Evaluate nuclear size relative to neutrophils (use as internal control)

• Assess chromatin pattern, nucleoli, and N:C ratio at high power

• Count mitotic figures in representative areas

• Note cell population homogeneity
  

2. Apply grading criteria:

• Determine if cells are predominantly small/mature or large/immature

• Assess nuclear features systematically

• Document mitotic activity

3. Provide clear conclusions:

• State grade when confident (high-grade vs. low-grade)

• Recommend confirmatory testing when uncertain

• Include differential diagnoses when appropriate

4. Communicate limitations:

• Note that cytology cannot fully replace histopathology

• Recommend immunophenotyping for complete characterization

• Indicate if the sample quality limits interpretation

Integration with Advanced Diagnostics

AI-Assisted Cytology

Emerging technologies, such as AI-powered platforms, are enhancing cytologic evaluation by:

• Standardizing assessment: Reducing inter-observer variability

• Quantifying features: Providing objective measurements of nuclear size, N:C ratio, and chromatin patterns

• Pattern recognition: Identifying subtle morphologic features that may predict grade

• Improving efficiency: Allowing rapid screening of samples

Complementary Testing

• Flow cytometry: Confirms monoclonality and provides immunophenotype

• PARR testing: Molecular confirmation of clonality

• Histopathology: Gold standard for definitive grading and subtyping

• Immunohistochemistry: Identifies specific cell lineage markers

Conclusion

Cytologic grading of canine lymphoma provides valuable prognostic information and guides treatment decisions. While high-grade lymphomas display aggressive morphologic features including large nuclear size, prominent nucleoli, and high mitotic activity, low-grade lymphomas are characterized by small, mature-appearing cells with condensed chromatin and minimal proliferative activity. Recognition of these key differences enables veterinarians to provide informed prognostic counseling and appropriate therapeutic recommendations. However, cytology should be integrated with clinical findings and, when possible, confirmatory testing to ensure accurate diagnosis and optimal patient management.

Key Takeaways

✓ High-grade lymphomas: Large cells, irregular nuclei, prominent nucleoli, high mitotic index

✓ Low-grade lymphomas: Small cells, condensed chromatin, inconspicuous nucleoli, low mitotic index

✓ Nuclear size comparison: Use neutrophils as internal reference (high-grade >1.5-2× neutrophil diameter)

✓ Clinical context matters: Integrate cytology with signalment, clinical presentation, and staging

✓ Know limitations: Recommend confirmatory testing when diagnosis is uncertain

✓ Communication is key: Clearly document confidence level and diagnostic caveats

Selected References

1. Camus MS, Priest HL, Koehler JW, et al. Cytologic criteria for myeloma cell identification. Vet Clin Pathol. 2016;45(4):572-588.

2. Poggi A, Miniscalco B, Morello E, et al. Flow cytometric evaluation of Ki67 for the determination of malignancy grade in canine lymphoma. Vet Comp Oncol. 2015;13(4):475-480.

3. Rout ED, Labadie JD, Yoshimoto SK, Avery PR, Avery AC. Optimization and diagnostic utility of immunocytochemistry on cytologic smears of canine and feline lymphomas. Vet Clin Pathol. 2018;47(4):586-598.

4. Marconato L, Frayssinet P, Rouquet N, et al. Randomized, placebo-controlled, double-blinded chemoimmunotherapy clinical trial in a pet dog model of diffuse large B-cell lymphoma. Clin Cancer Res. 2014;20(3):668-677.

5. Aresu L, Ferraresso S, Marconato L, et al. New molecular and therapeutic insights into canine diffuse large B-cell lymphoma elucidates the role of the dog as a model for human disease. Haematologica. 2019;104(6):e256-e259.

6. Comazzi S, Gelain ME, Martini V, et al. Immunophenotype predicts survival time in dogs with chronic lymphocytic leukemia. J Vet Intern Med. 2011;25(1):100-106.

7. Valli VE, Kass PH, San Myint M, Scott F. Canine lymphomas: association of classification type, disease stage, tumor subtype, mitotic rate, and treatment with survival. Vet Pathol. 2013;50(5):738-748.

8. Ponce F, Marchal T, Magnol JP, et al. A morphological study of 608 cases of canine malignant lymphoma in France with a focus on comparative similarities between canine and human lymphoma morphology. Vet Pathol. 2010;47(3):414-433.

9. Martini V, Poggi A, Riondato F, et al. Flow-cytometric detection of phenotypic aberrancies in canine small clear cell lymphoma. Vet Comp Oncol. 2015;13(3):281-287.

10. Seelig DM, Avery AC, Ehrhart EJ, Linden MA. The comparative diagnostic features of canine and human lymphoma. Vet Sci. 2016;3(2):11.