Cytology of lymphoid organs (Proceedings)

As the primary cell type present in lymphoid organs is lymphocytes, the first part of this handout will describe the morphologic features of lymphocytes and then briefly describe other methods to assess lymphocytes. The second part of the handout will describe the interpretation of various lymphoid organs.

Lymphocyte types

Small lymphocytes are smaller in size than a neutrophil and have a round nuclei that takes up the majority of the cell. The nuclei contain densely aggregated chromatin forming large chromocenters (condensed chromatin). Nucleoli are not seen. The cytoplasm is scant (sometimes only a very thin rim is visible) and lightly basophilic in color. These are typically called 'mature lymphocytes'. However, early lymphoid progenitor cells, hematopoietic stem cells, certain stages and types of dendritic cells, and other immature precursor cells may have a very similar morphology to 'mature, well-differentiated, small, resting lymphocytes'.

Intermediate to large lymphocytes range in size from slightly larger than small lymphocytes to the size of neutrophils. The nuclei still takes up the majority of the cell, however more abundant cytoplasm is visible in these cells. Often, the nuclei is placed eccentrically within the cytoplasm. The nuclear chromatin is finely clumped to granular. Typically, nucleoli are not seen although strands of loosely clumped nuclear chromatin may be mistaken for nucleoli. The cytoplasm is lightly basophilic in color. Occasionally these cells contain azurophilic granules suggestive of a natural killer (NK) phenotype.

Lymphoblasts are as large as a neutrophil or larger. Size alone does not indicate neoplasia. Very large lymphoblasts (2-4x the size of neutrophils) may be seen in reactive and hyperplastic processes. Lymphoblasts contain round to oval nuclei with fine or stippled chromatin (loosely aggregated chromatin). One or more nucleoli may be visible. The cytoplasm is moderately to deeply basophilic. Occasionally (seen more in cats than dogs) the cytoplasm may contain punctate vacuoles.

Reactive lymphocytes are similar in morphology to small lymphocytes but are slightly larger and have more abundant, more basophilic cytoplasm.

Plasma cells are intermediate sized cells that contain small, round, eccentrically placed nuclei with condensed chromatin. Cytoplasm is abundant, deeply basophilic, and often contains a prominent, eccentric, perinuclear, clear zone that corresponds to the Golgi.

Morphologic features and typical findings used to characterize atypical lymphocytes

Lymphoglandular bodies are round, homogeneous, basophilic structures comprised of cytoplasmic fragments. The presence of lymphoglandular bodies is seen in cytologic preparations of lymphoid tissue that contains increased numbers of lymphoblasts. This can be due to neoplasia (lymphoma) or hyperplasia.

The presence of an eccentric, perinuclear clearing zone is often suggested as a feature of B-cells and plasma cells. The clearing zone is the Golgi and it is a prominent feature in plasma cells. However, the Golgi apparatus is an organelle found in most cells, including T-cells and myeloid cells.

In humans, Sezary cells are described as medium to large lymphocytes with ceribriform nuclei. These neoplastic T-cells are characteristic features of Sezary syndrome which encompasses mycosis fungoides, an epitheliotropic variant of cutaneous lymphoma. A similar syndrome occurs in dogs but has been rarely reported in cats. In dogs, epitheliotropic T-cell lymphoma is also seen in the gastrointestinal tract. T-cells predominate in both the cutaneous and GI variants. Interestingly, expression of protein gene product 9.5 (PGP 9.5), a marker previously considered specific for neural and neuroendocrine tissues, was recently detected in over 8/14 cases of canine cutaneous mycosis fungoides suggesting that there may be other biologic differences between the human and canine variants.

In humans, the presence of flower cells or cloverleaf cells is most often associated with T-cell disease and is particularly a feature of infection with human T-lymphotrophic virus-1 (HTLV-1). In dogs and cats, similar morphology has been seen in both B-cell and T-cell lymphoproliferative disease as well as myeloproliferative disease.

In humans, CLL is considered a disease of B-cells. In contrast, CLL of dogs and cats is primarily a T-cell disease. In dogs, CD8+ (cytotoxic) CLL predominate while in cats, CD4+ (T-helper) CLL is more common. However, there is variation in the disease in both dogs and cats and B-cell, CD4+ T-cell, and CD8+ T-cell CLL have all been diagnosed in small animals.

Chronic lymphocytosis: In dogs, a chronic lymphocytosis comprised of intermediate sized lymphocytes with small azurophilic granules has been reported in association with Ehrlichiosis.

Flow cytometry

Flow cytometric analysis is used to define cells through a panel of phenotypic markers and receptors (usually surface) and provide a more objective characterization of these abnormal lymphocyte populations. Lymphocyte phenotyping by flow cytometric analysis has become an established diagnostic assay for assessment of abnormal hematopoietic populations in small animal patients. In both dogs and cats, a number of well-characterized antibodies are available for evaluation of lymphoid populations with fewer antibodies available for examination of histiocytic, myeloid, erythroid, and megakaryocytic cells.

Clonality

There are no specific phenotypic markers that identify a clonal T-cell population in humans or veterinary species. In humans, the use of kappa and lambda light chain markers can help to identify clonal B-cell populations when the populations uniformly express one or the other light chain. However, there are currently no antibodies for detection of canine or feline light chains by flow cytometry. In addition, lambda light chains predominate over kappa chains in dogs, reducing the utility of light chain detection. Therefore, identification of T-cell or B-cell clonality in dogs and cats requires detection of receptor gene rearrangement. This is most typically done by PCR and is referred to as PCR for antigen rearrangement (PARR). As part of their development, T-cells undergo rearrangement of genes encoding the T-cell receptor (TCR) while B-cells undergo rearrangement of genes encoding the immunoglobulin (Ig) receptor. The result is that nearly every lymphocyte in the body has a unique TCR or Ig receptor. PARR of normal tissue detects a smear or ladder of PCR products representing the diversity of the normal receptors. Because neoplastic transformation typically occurs after the cells have undergone receptor rearrangement, all malignant daughter cells will have the same antigen receptor gene. This is detected on PCR as a single band and represents a monoclonal population. Occasionally bi or tri-clonal populations may also be detected.

Cytology of lymph nodes

In a reactive/hyperplastic lymph node, small lymphocytes predominate although the lymphoid population is heterogeneous with increased numbers of plasma cells, medium lymphocytes, and lymphoblasts. Macrophages, neutrophils, eosinophils, and mast cells may also be increased. Mandibular and mesenteric lymph nodes are often "normally reactive" due to drainage of the oral cavity and intestine.

Lymphadenitis is characterized by increased numbers of inflammatory cells. The cell type varies with the type of inflammation. Examination of lymph nodes aspirates is also useful for diagnosing thoracic and abdominal lesions. Lymph nodes draining inflammatory lesions also serve as a site for accumulation of organisms.

• Increased neutrophils (>5%) = suppurative (purulent) inflammation * if blood contamination present, need to consider the peripheral neutrophil count when evaluating for inflammation.

• Epithelioid macrophages = granulomatous inflammation

• Increased macrophages and neutrophils = mixed to pyogranulomatous inflammation

• Increased eosinophils = eosinophilic inflammation

Lymphoma is relatively easy to diagnose when there is an increased percentage of immature forms. Often these will present with >80% to a monomorphic population of lymphoblasts or intermediate lymphocytes. Small cell lymphoma and lymphoma of intermediate cells can be very difficult to diagnose by cytology. If medium or small cell lymphoma is present, you can look for a monomorphic population, no progression or differentiation, and subtle signs of atypia. However, these usually require examination of histologic architecture. Because of the rapid proliferation and increased turn-over seen in lymphoma, especially lymphoblastic lymphoma, macrophages and lymphoglandular bodies are common.

Non-lymphoid tissue can be obtained in two scenarios. The first is when the lymph node contains metastatic neoplasia. Atypical cells representing metastatic neoplasia are often more easily identified and characterized within the lymph node environment than in the mass lesion itself where inflammation, necrosis, and reactive tissue hyperplasia can confuse the diagnosis. The lymph node may be effaced in this process. The second is when the lymph node is not aspirated (e.g. fat, salivary gland, muscle). The latter is relatively common and should be excluded before considering effacement of the lymph node.

Spleen

Normal spleen contains primarily blood and lymphoid cells. Occasionally, a few monomorphic spindled cells representing fibrocytes or endothelial cells are seen. Lymphocytes are evaluated similarly to those seen in lymph nodes. Most of the lymphoid cells are small lymphocytes with a lesser population of prolymphocytes and lymphoblasts. Occasional plasma cells, macrophages, and mast cells may also be present.

Reactive hyperplasia is identified through the detection of increased numbers of prolymphocytes, lymphoblasts, and plasma cells. A marked plasmacytosis, resembling multiple myeloma, may be seen in canine Ehrlichiosis, leishmaniasis, and feline immunodeficiency infections.

Extramedullary hematopoiesis (EMH) is commonly seen cytologically in splenic aspirates. Erythroid precursors and megakaryocytes are seen more frequently, with granulocytic EMH less common. Immature cells are easily confused with lymphoblasts and can lead to the erroneous diagnosis of lymphoma. Visualization of erythroid progression (including hemoglobinization) is helpful for differentiating erythroid and lymphoid precursors.

Lymphoma is characterized by a monomorphic population of lymphoid cells. Typically, these are lymphoblasts, however occasionally prolymphocyte or small cell variants are seen which require histologic evaluation of splenic architecture. Extraskeletal multiple myeloma and plasmacytoma may be seen in both the spleen and the liver. These are characterized by a population of plasma cells. Well-differentiated plasma cells have eccentric nuclei, clumped "cartwheel" chromatin, deeply basophilic cytoplasm, and a paranuclear clear area. Binucleate cells and nuclei with less clumped chromatin may be seen. Poorly differentiated plasma cells need to be differentiated from amelanotic melanoma, osteosarcoma, and malignant histiocytosis.

Myeloproliferative disease involving any of the cell lines or the stem cells may involve the spleen and must be differentiated from EMH. Generally, large numbers of a relatively monomorphic population of cells are seen in cases of myeloproliferative disease. Concurrent evaluation of peripheral blood and bone marrow by special stains is often necessary to fully characterize the neoplastic process.

Splenic mast cell tumors are common, particularly in cats.

Primary splenic neoplasia such as hemangiosarcoma is difficult to diagnosis by aspiration cytology, but some of the more solid hemangiosarcomas exfoliate well resulting in large numbers of atypical mesenchymal cells. However most aspirates of hemangiosarcomas result in blood and only rare spindled cells.

Thymus

Thymoma, lymphoma, and other primary and metastatic neoplasia may appear as anterior mediastinal masses. Thymoma is characterized by numerous small lymphocytes, a variable proportion of thymic epithelial cells (the neoplastic population), and occasional mast cells. By flow phenotyping, a significant proportion of the lymphocytes will be CD4+CD8+ double positive thymic precursors that helps to differentiate thymoma from lymphoid neoplasia. Mediastinal lymphoma appears similar to lymphoma at other sites.

Lymphoid leukemia

Acute leukemia in the dog appears slightly more likely to be of myeloid than lymphoid origin, and about 10% of acute leukemias lack identifiable differentiation markers (acute undifferentiated leukemia). Acute lymphoid leukemia (ALL) in the dog may be comprised of cells of B, T, or NK origin. CD34 is often detected on acute myeloid leukemia (AML) and ALL, but is rarely (or not) detected on lymphoma and therefore serves as a useful marker to differentiate ALL with tissue involvement from lymphoma with marked leukemia.

In dogs, chronic lymphocytic leukemia (CLL) appears to be primarily a T-cell disease although B-cell CLL has also been reported (3:1 ratio of T:B). The cytologic morphology of T-cell CLL in dogs is typically of granular lymphocytes. The granular cells primarily express CD3, CD8, and CD11d. Detection of the alpha/beta TCR is more common although about a third of the reported cases express the gamma/delta TCR. Non-granular T-cell CLL more commonly express the alpha/beta TCR but may be either CD4 or CD8 positive. B-cell CLL in dogs express CD21 and/or CD79a. The majority (~95% of those examined), also express CD1c. CD5 (which is commonly seen in human B-cell CLL) is not detected on canine B-cell CLL. B-cell CLL and T-cell CLL appear to have somewhat different patterns of disease progression. B-cell CLL affects the bone marrow early in disease and may be considered a primary bone marrow disease. T-cell CLL typically does not affect the bone marrow until late in the disease and may spread from the marrow after splenic involvement.

Similar to that seen in dogs, feline CLL is primarily a T-cell disease. However, unlike that seen in dogs, feline CLL is primarily a result of CD4 or helper T-cell proliferation although occasional cases of CD8+, CD4CD8 double positive, and CD4CD8 double negative CLL have also been reported.