CHAPTER 11 The Lymph Nodes

ARCHITECTURE

When interpreting a cytological specimen of the lymph node, it is useful to keep in mind the histological structure and different cell types that are found in this tissue. The node is composed of a capsule, cortex, medulla, and sinuses (subcapsular, cortical, and medullary).¹ The cortex or more peripheral area of the node is divided into follicular and diffuse (parafollicular cortex or paracortex) regions, and the medulla or more central area, into the medullary cords and sinuses (Figure 11-1).

Figure 11-1 The lymph node has two basic parts, the cortex and the medulla. The cortex has both follicular and diffuse or parafollicular regions. The parafollicular region gradually transforms into medullary cords of B-lymphocytes and plasma cells, which are surround by sinuses containing macrophages attached to reticular fibers. Different populations of lymphyocytes in these areas and other cells are found in nodal aspirates. (H&E stain.)

Within the parafollicular cortex are high endothelial venules through which both B- and T-lymphocytes from the blood enter the node. This region is also rich in interdigitating reticulum cells (IDCs), a specialized antigen-presenting cell. The initial immune response requires that the antigen presented by IDCs be recognized by T-lymphocytes and early B-lymphocytes in the parafollicular cortex, whereas the differentiation of B-lymphocytes in response to antigen occurs in the follicular cortex.

Follicles contain predominantly B-lineage lymphocytes. The primary follicles are composed of small, dark-staining lymphocytes. In contrast, secondary follicles have a peripheral rim or mantle zone of small dark lymphoid cells similar to those in primary follicles and a central germinal center. In the germinal center, specialized cells of the mononuclear phagocytic system (MPS), the follicular dendritic cells (FDCs) capture antigen on their surfaces to promote B-lymphocyte differentiation. Thus, small resting B-cells undergo mitosis and divide to become the larger, more irregular small-cleaved, intermediate and large blast cells in the germinal center of a reactive node (follicular hyperplasia). T-cells (mainly CD4₊ helper cells) that play a role in stimulating B-cells are also found in the follicles. Surviving B-cells may eventually differentiate into plasma cells, migrating to the medullary cords or leaving the node.

The parafollicular zone of the lymph node gradually transforms into medullary cords that are populated by B-cells and plasma cells. Sinuses containing macrophages surround these cords. A reactive process in the lymph node may also result in hyperplasia of the parafollicular region, of sinus cells (sinus histiocytosis), or of plasma cells (plasma cell hyperplasia). These different types of hyperplasia may occur by themselves or in combination.

Lymph nodes are strategically located at sites throughout the body and are involved in a variety of local and systemic disease processes. Antigen reaches the node via the afferent lymphatics. The lymph percolates through the sinus and sinusoidal walls into the parenchyma (Figure 11-2), where foreign substances (antigens) are taken up and processed by specialized cells of the MPS. The sinuses (subcapsular, cortical and medullary) form a network of branching channels that converge at the hilus of the node to exit by the efferent lymphatics. The primary functions of the lymph nodes include filtering particles and microorganisms, exposing antigens to circulating lymphocytes, and activating B- and T-lymphocytes. The superficial, subcutaneous location of some lymph nodes (mandibular, superficial cervical, inguinal, and popliteal) allows easy detection of enlargement and access for fine-needle aspiration (FNA) cytology. It is appropriate to aspirate any node that is enlarged, and in the case of lymph nodes draining areas affected by neoplasia, even in the absence of enlagement, aspiration may be justified.²

Figure 11-2 Lymph enters the node via the afferent lymphatics, percolating through subcapsular sinuses and sinusoids, where foreign substances (antigens) are taken up and processed. The secondary follicles in this node have a peripheral rim or mantle zone and a pale-staining, central germinal center. The differentiation of B-lymphocytes in response to antigen occurs in the germinal center of the follicular cortex. (H&E stain.)

GENERAL CONSIDERATIONS

Lymph node aspiration cytology has become a popular procedure in human medicine in recent years, because of its great convenience.³ Similarly, this high-yield diagnostic technique is frequently used in veterinary medicine.⁴^–⁷ There are a few points to consider when obtaining nodal samples for cytological evaluation.

A normal lymph node is small and often difficult to aspirate. It is not uncommon for the cytology of a normal node to contain mostly perinodal adipose tissue and only a few or no lymphocytes. If multiple nodes are enlarged, then sampling from several nodes is recommended. Because the submandibular nodes drain the oral cavity, they often become enlarged, reactive, and/or inflamed. A confusing mixture of malignant, reactive, and inflammatory cells may limit the accuracy of cytologic diagnosis of lymphoma based on an FNA of these nodes. Thus, sampling of submandibular nodes should be avoided in cases where there is generalized lymph node enlargement. The prescapular and popliteal nodes are often a better choice. The mandibular salivary gland is not uncommonly mistaken for a node and aspirated. However the presence of large, foamy epithelial cells either individually or in clusters and mucus in the background allows the tissue to be easily identified.

Consideration also should be given to the size of the lymph node when deciding which node to aspirate — very large nodes may have areas of hemorrhage or necrosis. If the node must be sampled, the needle should be directed tangentially, avoiding the more central portions.⁸ Finally, when obtaining a sample for cytological evaluation, it is important to remember that the lymph node is a heterogenous tissue, and multiple areas within the node should be sampled to be certain that what you have obtained is representative. While keeping the needle in the node, repeatedly advance and withdraw the needle in multiple directions until a small amount of aspirate appears in the hub of the needle. This procedure can be done using a syringe to apply gentle suction or with only the needle. If the former technique is used, release the suction before removing the needle from the node. Overly vigorous aspiration of the lymph node can produce significant hemodilution and cells can rupture, limiting the interpretation of your sample. A large volume of aspirate is not required; the material within the hub of your needle is sufficient for making cytologic preparations. Because the lymphocytes are fragile, care also must be taken to apply only minimal pressure when making slide preparations to prevent excessive rupturing of cells. The slides are air-dried (not heat-fixed) and stained for evaluation.

FINE-NEEDLE ASPIRATION

FNA is a relatively safe and painless procedure, allowing for rapid and inexpensive sampling of peripheral lymph nodes. It does not require hospital admission or anesthesia of the pet. The role of this procedure is summarized in Box 11-1.

Box 11-1 The Role of Fine-Needle Aspiration Cytology of Lymph Nodes

1. Diagnosis of infectious disease.

2. Diagnosis of hyperplasia or reactive lymphadenopathy and recognition of specific conditions (i.e., lymph node hyperplasia of young cats). If a cause for the change is not apparent and/or resolution does not occur, follow-up and subsequent biopsy is indicated.

3. Diagnosis of metastatic neoplasia and indication of possible primary site.

4. Diagnosis of lymphoma that is optimally followed by a biopsy for confirmation and accurate subtyping.

5. If known malignancy, such as lymphoma or a metastatic mast cell tumor, staging and monitoring for relapse or effects of chemotherapy.

6. Allows sampling of multiple sites as well as obtaining samples from surgically inaccessible sites or from medically unfit patients.

7. Obtaining material for clonality and research studies.

CYTOLOGIC FINDINGS

Normal Lymph Node

In the absence of architectural features that can be appreciated in a histologic section of a lymph node, the interpretation of cytology relies on proportions of different cell types and an understanding of what proportions are normal versus abnormal for these cell types. Small, well-differentiated lymphocytes compose greater than 75% to 85% of the total nucleated cell population (Figures 11-3 through 11-7).⁴^–⁷ They have round nuclei that are about 1.0 to <1.5 times the size of a mature red blood cell (RBC) with an overall cell size that is smaller than that of a neutrophil. Their chromatin is densely clumped and nucleoli are not visible. The nuclear-to-cytoplasmic ratio is high with a narrow rim of basophilic cytoplasm. In addition to small lymphocytes, a normal node should have low numbers (<10% to 15%) of lymphocytes that are intermediate to large (often called lymphoblasts) in size; their nuclei are 1.5 to 3 times the size of an RBC with an overall size ranging from about that of a neutrophil or larger to up to 4 times the size of an RBC (see Figure 11-5). Their chromatin is less clumped and nucleoli may be visible and even multiple and/or prominent. The cytoplasm is pale blue and more abundant than in small lymphocytes.

Figure 11-3 Aspirate from a hyperplastic lymph node. Small lymphocytes (arrows) and plasma cells characterize the reaction. Note that small lymphocytes are smaller than neutrophils and their nuclei are about the size of a red blood cell. Many free nuclei, identified by pink, homogeneous chromatin and an absence of cytoplasm, are evident. (Wright’s stain.)

Figure 11-4 Small lymphocytes, plasma cells, and a large transformed lymphocyte (arrow) characterize this aspirate from a hyperplastic lymph node. Irregular, pink nuclei from lysed cells are seen. (Wright’s stain.)

Figure 11-5 Plasma cells, small lymphocytes, and two large lymphocytes (lymphoblasts) characterize this aspirate from a hyperplastic lymph node. The plasma cell with the vacuolated cytoplasm is a Mott cell containing Russell bodies (arrow). The pink, amorphous structures are free nuclear material. (Wright’s stain.)

Figure 11-6 Small lymphocytes, plasma cells, neutrophils, and a single mast cell are present in this aspirate from a hyperplastic lymph node. (Wright’s stain.)

Figure 11-7 Seen among small and medium-sized lymphocytes is a large macrophage containing phagocytized debris. (Wright’s stain.)

Plasma cells have small, round, eccentric nuclei with condensed chromatin (see Figures 11-3 through 11-6). Their abundant cytoplasm is deep blue and has a prominent, clear Golgi zone. Immature plasma cells (transformed B-lymphocytes) are larger and have less aggregated chromatin and a higher nuclear-to-cytoplasmic ratio (see Figure 11-4). Their very blue cytoplasm may contain discrete vacuoles. Plasma cells in various stages of development are seen in small numbers in normal lymph nodes, but typically represent <3% of the total nucleated cell population.

Macrophages, characterized by abundant cytoplasm often containing vacuoles and granular debris, also are found in small numbers (see Figure 11-7). Macrophages from areas of intense lymphopoiesis and cellular turnover may contain prominent basophilic nuclear debris (tingible bodies) (Figure 11-8). Occasionally, small numbers of neutrophils, eosinophils, and mast cells are observed in a normal node (see Figure 11-6). Each of these cell types should represent less than 1% of the cell populations in a normal node. It is important to consider the amount of blood contamination when making this assessment. Reticular cells and endothelial cells are common in lymph nodes, but these tissue-bound cells are rarely aspirated intact. They usually appear as large swollen nuclei, often devoid of cytoplasm (Figure 11-9).