Cellular morphology and cytochemistry are the traditional methods to characterize and classify hemolymphatic neoplasms. Lymphomas may be subclassified as B or T cell type or distinguished from nonlymphoid populations. Cellular phenotyping in hematopoietic neoplasms has been found to not only guide therapeutic decisions but provide prognostic information in both human and veterinary medicine.

Flow cytometry is a rapid, sensitive, quantitative method of automated cell analysis. In the veterinary clinical setting, flow cytometry is used primarily to determine cellular phenotypes in hemolymphatic disorders. Cellular phenotypes by flow cytometry are determined by using monoclonal antibodies tagged to fluorochromes directed against surface molecules expressed on leukocytes. Another technique to evaluate cell phenotype is the use of immunocytochemistry that can be applied to affected organ aspirates, impressions and cytospin preparations. This is a routine protocol performed at many academic and commercial laboratories that involves application of a specific, monoclonal primary antibody often followed by application of a biotinylated secondary antibody, streptavidin-horseradish peroxidase binder and aminoethylcarbazole or benzidine colorant. The advantage of immunocytochemistry is the ability to use routine diagnostic samples and see the specific cells that are stained. Additionally, not all antibodies can be applied to histologic specimens so immunocytochemistry allows more in-depth identification of cell types than is possible by immunohistochemistry.

There are several antibodies available to phenotypically characterize lymphomas or lymphoid leukemias (Table 1). When applied as a panel, multiple characteristics become apparent which relate to the biologic nature of the lymphocyte. Positive neoplastic cell staining for any combination of CD21, BLA 36, CD 79_alpha, and immunoglobulins can be interpreted as B-cell immunophenotype. Positive neoplastic cell staining for any combination of CD3, CD3^epsilon, CD4, and CD8_alpha can be interpreted as T-cell immunophenotype.¹

Table 1. List of antibodies for immunophenotyping in the dog and cat.

Classification of Lymphoma

Once the determination of immunophenotype is made the information is correlated with anatomic disease presentation plus the cytologic and histologic characteristics of the lymphoid cells to classify the condition into one of the categories from the WHO scheme (Table 2) as described in the 2001 WHO publication.² Placement of the condition into a disease category provides prognostic information as well as understanding about the biologic behavior of the disease. The relevance of the WHO scheme has been examined in a number of canine cases (Table 3) and found to be clinically useful.³

Table 2. Recognized subtypes for canine lymphoid malignancies using WHO classification scheme.

Table 3. Median survival times from 49 Purdue canine cases.

Classified by WHO Disease Entities with a or b clinical substage

References

1.  Wilkerson M J. Lineage differentiation of canine hematopoietic disorders: Lessons from the human side, in Proceedings. 22nd Annu Meet ACVIM Forum 2004; 625-627.

2.  Jaffe ES et al.World Health Organization Classification of Tumours: Pathology and Genetics of Tumours of Haematopoietic and Lymphoid Tissues. Lyon, France: IARC Press, 2001;109-271

3.  Raskin RE, Fox LE. Clinical relevance of the World Health Organization classification of lymphoid neoplasms in dogs. Vet Pathol 2003; 40: 5