(Immuno)phenotyping: What, why, and how? A case report and discussion

WHAT and WHY: Immunophenotyping employs antibodies to label antigens (proteins) on or within cells that may characterize or identify a cell or tissue type. In clinical veterinary medicine, we commonly phenotype lymphoid tumor cells to determine if they are of T cell or B cell origin, for two reasons. For decades, we have been phenotyping canine lymphomas to more accurately assess prognosis. In a recent report, three subtypes have been identified with very different prognoses: low-grade T cell lymphoma, high-grade T cell lymphoma, and B cell lymphoma.1 Generally speaking, the median survival time of a canine high-grade B cell lymphoma patient that responds well to multi-agent chemotherapy is 10-12 months, versus 5-6 months for a high-grade T cell lymphoma patient receiving similar treatment. Furthermore, with the advent of canine-specific immunotherapies developed to specifically target malignant T cell and B cells, a second reason to determine the phenotype of lymphoid malignancies has recently emerged.

HOW: In veterinary medicine, we currently use three methods to (immuno)phenotype lymphoid malignancies, which are IHC, ICC, and flow cytometry. These methods commonly use antibodies directed at CD3 on the surface of T cells, and CD20, CD21, or CD79a on or within B cells. For these methods to work, the cells of interest have to express a consistent pattern of positivity and negativity for an array of CD markers. For example, a T cell lymphoma will typically be CD3 positive and CD79a negative, while a B cell lymphoma will be CD3 negative and CD79a positive.

Another method used to phenotype lymphoid malignancies that does not use antibodies (hence, this is not true immunophenotyping), is PARR. This PCR method employs DNA primers to amplify regions of the B cell receptor (BCR) genes or T cell receptor (TCR) genes, to detect the presence of a clonal T or B cell population.

Method PROS CONS
Immunohistochemistry (IHC) Architecture of tumor preserved, pathologists can see cellular arrangements of T cells and B cells Biopsy required (sedation, surgery, costs)
Immunocytochemistry (ICC) Performed on air-dried, cytology slides (can be run months to years after slides are made) Requires unstained slides with ample, intact cellularity

Limited number of antibodies for antigen detection

Flow Cytometry Can employ a long list of antibodies for detecting many cellular markers Requires well-preserved cells in a fluid medium (sterile saline, blood), so must be run within days of acquisition
PCR for Antigen Receptor Rearrangement (PARR) Requires DNA, not intact cells, so can be run on stained cytology slides, biopsy specimens, and fluid ~20% of canine samples with confirmed malignancy will give a negative result


CASE:

A 4-year-old, spayed female French Bulldog presented to a referral hospital with a cytologic diagnosis of lymphoma and enlarged mandibular lymph nodes. The patient received prednisone with asparaginase treatment and exhibited some response, but did not achieve a full clinical remission. Additional aspirates were obtained and submitted for flow cytometric evaluation. The cells were negative for all standard T cell and B cell markers (CD3, CD20, etc.), but were CD34+ and CD14+, suggesting myeloid origin.

Additional fine needle aspirates of the enlarged mandibular lymph nodes were obtained and submitted for a second cytologic evaluation. The slides were highly cellular and consisted predominantly of intermediate to large immature round cells (lymphocytes), with few small lymphocytes, and occasional neutrophils, mast cells, and plasma cells. The intermediate to large lymphocytes consisted of a small amount of basophilic cytoplasm, a small perinuclear clear area, and an intermediate to large round nucleus with a finely-stippled chromatin pattern and indistinct nucleoli. Occasional mitotic figures are noted. The background consists of many lymphoglandular bodies and occasional free nuclei. The cytologic findings were again consistent with high-grade lymphoma. See photo below.

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Since these cells did not express typical lymphoid markers via flow cytometry, the cytology slides were sent to an academic veterinary diagnostic laboratory for PARR analysis. The BCR (B cell receptor) PARR gel showed a crisp band that was not present on the TCRγ PARR gel, confirming a clonal population of B lymphocytes thus consistent with a diagnosis of B cell lymphoma. As a potential explanation for the negative flow cytometry results, hypothetically, these neoplastic B cells may have been too immature to express typical B cell antigens (CD20, CD21, CD79a). In this case, the PARR assay results helped further confirm the original diagnosis of lymphoma and provided the B cell phenotype, and thus providing the clinician the necessary information to proceed with confidence toward a multi-agent chemotherapy protocol for canine lymphoma.

Casey J. LeBlanc, DVM, PhD
Diplomate, ACVP (Clinical Pathology)
Eastern VetPath
Springfield, VA
[email protected]

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References:

  1. Molecular profiling reveals prognostically significant subtypes of canine lymphoma. Frantz AM, et al. Vet Pathol 2013 Jul;50(4):693-703.
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