Enlarged lymph nodes: Differentiating reactive lymphadenitis, tuberculous lymphadenitis, and lymphoma through anatomical pathology

1. What are Lymph nodes and when are they considered enlarged?

1.1. Structure and function of Lymph nodes

Lymph nodes are organized lymphoid organs containing lymphocytes within a delicate reticular stroma [2]. A lymph node consists of the cortex, which contains B cells arranged in lymphoid follicles and germinal centers; the paracortex, which contains T cells; and the medullary sinuses and medullary cords [1]. The primary functions of lymph nodes are to filter fluid and tissue components and to provide sites for lymphocyte production and the immune functions necessary for normal physiological processes [1].

1.2. Definition of Lymphadenopathy

Lymphadenopathy refers to lymph nodes that are enlarged beyond their normal size. Although lymph node size varies according to anatomical location and overall health, lymph nodes are generally considered enlarged when their diameter exceeds 1 cm at most sites, 0.5 cm in the supraclavicular region, or 1.5 cm in the inguinal region [n]. Lymphadenopathy may represent a benign response to infection or inflammation, but it can also indicate more serious conditions, such as tuberculous lymphadenitis or malignancies, including lymphoma and metastatic cancer [1].

2. Major causes of enlarged Lymph nodes

Lymphadenopathy can result from various causes. Three important conditions that must be differentiated are reactive lymphadenitis, tuberculous lymphadenitis, and lymphoma.

2.1. Reactive Lymphadenitis

Reactive lymphadenitis is the enlargement of lymph nodes in response to an immune stimulus, such as a bacterial or viral infection, inflammation, or tissue injury [1]. It is a benign response that indicates the immune system is working to combat the triggering condition.

2.2. Tuberculous Lymphadenitis

Tuberculous lymphadenitis is a form of extrapulmonary tuberculosis caused by Mycobacterium tuberculosis that affects the lymph nodes. It is one of the most common forms of extrapulmonary tuberculosis and frequently affects the cervical lymph nodes [n].

2.3. Lymphoma

Lymphoma is a cancer that originates in lymphocytes, a type of white blood cell that plays an important role in the immune system. When lymphocytes grow abnormally and uncontrollably, they can accumulate in lymph nodes and other organs, causing lymphadenopathy and other symptoms [3]. Lymphoma is broadly divided into two main groups: Hodgkin lymphoma and non-Hodgkin lymphoma [3].

2.4. Other causes

In addition to these three conditions, lymphadenopathy may result from autoimmune diseases, metastatic cancer originating in other organs, or other less common conditions [1].

3. Anatomical pathology in the differential diagnosis of enlarged Lymph nodes

Anatomical pathology, particularly the examination of hematoxylin and eosin (H&E)-stained tissue sections, is an important initial diagnostic method for differentiating the causes of lymphadenopathy [2].

3.1. Tissue preparation and hematoxylin and eosin (H&E) staining

Lymph node tissue is usually obtained through biopsy, either by excisional biopsy or core needle biopsy. The specimen is then fixed, processed, embedded in paraffin, and cut into thin sections. These sections are stained with H&E for microscopic examination [1]. H&E staining clearly demonstrates tissue architecture, cellular morphology, and pathological changes.

3.2. Histopathological features of reactive Lymphadenitis

In reactive lymphadenitis, the overall lymph node architecture is generally preserved, although different components may proliferate depending on the type of immune stimulus [2].

• Follicular hyperplasia: This is the most common pattern, characterized by an increase in the size and number of secondary lymphoid follicles, with enlarged, pale germinal centers containing large lymphoblasts and tingible body macrophages [1, 2]. The mantle zone surrounding each germinal center is usually thin and well defined [1].
• Paracortical hyperplasia: This is characterized by proliferation of T lymphocytes in the paracortex, sometimes accompanied by immunoblasts [2].
• Sinus histiocytosis: This refers to an increase in macrophages within the lymph node sinuses, particularly the subcapsular and medullary sinuses. These macrophages may contain pigments or cellular debris [1].

3.3. Histopathological features of tuberculous Lymphadenitis

Tuberculous lymphadenitis has characteristic histopathological features that help distinguish it from reactive lymphadenitis and lymphoma.

• Caseating granulomas: This is the most characteristic finding in tuberculous lymphadenitis. Granulomas consist of epithelioid cells and Langhans giant cells surrounding an amorphous, pale pink area of caseous necrosis [n].
• Langhans giant cells: These are multinucleated giant cells in which the nuclei are arranged in a horseshoe or circular pattern near the cell periphery [n].
• Lymphocytes, plasma cells, and fibrosis may also be present around the granulomas [n].
• Special staining, such as Ziehl–Neelsen staining, may help detect acid-fast bacilli associated with tuberculosis.

3.4. Histopathological features of Lymphoma

Lymphoma is diagnosed based on alterations in lymph node architecture and the morphology of abnormal lymphocytes [3].

• Disruption of normal lymph node architecture: In lymphoma, the normal architecture of the lymph node, including the cortex, paracortex, and medulla, is often partially or completely replaced by proliferating malignant lymphocytes [1, 3].
• Atypical lymphocytes: Lymphoma cells often exhibit a monomorphic appearance, with abnormal size and shape, irregular nuclei, prominent nucleoli, and increased mitotic activity [1, 3].
• Hodgkin and non-Hodgkin lymphoma:
  • Hodgkin lymphoma: Characterized by Reed–Sternberg (HRS) cells within an inflammatory background containing lymphocytes, plasma cells, eosinophils, and macrophages [3]. HRS cells are large and may have multiple nuclei or multilobated nuclei with prominent nucleoli [3].
  • Non-Hodgkin lymphoma: This group includes a wide variety of subtypes classified according to cellular morphology, growth pattern (diffuse or follicular), and immunophenotypic characteristics [3]. For example, diffuse large B-cell lymphoma (DLBCL) is characterized by large, atypical B lymphocytes that proliferate in a diffuse pattern [3].
  • A “starry-sky” appearance may be observed in certain lymphomas, particularly Burkitt lymphoma, due to macrophages containing phagocytosed cellular debris among the proliferating tumor cells [1].

4. Immunohistochemistry (IHC)

Immunohistochemistry (IHC) is an indispensable tool in the differential diagnosis of lymphadenopathy, particularly when routine histopathological examination alone is insufficient to establish a definitive diagnosis [3].

4.1. Principles and importance of IHC

IHC uses antigen–antibody interactions to detect specific proteins in cells [3]. Its advantages include cost-effectiveness, relatively rapid processing, and compatibility with formalin-fixed, paraffin-embedded tissue specimens [3]. IHC helps determine cell lineage (B cells or T cells), assess cellular maturation, identify specific genetic abnormalities through relevant markers, and identify potential therapeutic targets [3].

4.2. Common immunohistochemical markers used for differentiation

Immunohistochemical markers are used to identify cell lineage and classify lymphomas [3].

• B-cell markers:
  • CD20: A general B-cell marker expressed in most mature B-cell lymphomas [3].
  • CD79a: A useful alternative to CD20, particularly when CD20 expression is lost because of plasma cell differentiation or rituximab treatment [3].
• T-cell markers:
  • CD3: A general T-cell marker expressed in most mature T-cell and natural killer (NK)/T-cell lymphomas, although expression patterns vary by subtype [3].
  • CD5: A T-cell marker that may also be expressed in certain B-cell lymphomas, such as mantle cell lymphoma and chronic lymphocytic leukemia/small lymphocytic lymphoma (CLL/SLL) [3].
• Other markers:
  • CD10: Expressed in germinal center cells and may be present in follicular lymphoma and certain cases of diffuse large B-cell lymphoma [3].
  • CD15 and CD30: Commonly positive in classical Hodgkin lymphoma and certain anaplastic large cell lymphomas [3].
  • Ki-67: Reflects the proliferative activity of tumor cells [3].
  • Cyclin D1: A highly useful marker for mantle cell lymphoma [3].
  • SOX11: May help diagnose mantle cell lymphoma in cases without Cyclin D1 expression [3].
  • EBV in situ hybridization (EBV ISH): Detects Epstein–Barr virus and is important in diagnosing extranodal NK/T-cell lymphoma, nasal type, and certain EBV-positive large B-cell lymphomas [3].

4.3. Application of IHC in diagnosing reactive lymphadenitis

In reactive lymphadenitis, IHC can help support the benign, polyclonal nature of the lymphocyte population, reflecting the proliferation of both B cells and T cells [2]. Enlarged lymphoid follicles typically express CD20 in B cells, while the paracortex expresses CD3 in T cells. These findings generally show an appropriate distribution without evidence of an abnormal imbalance in cell populations [2].

4.4. Application of IHC in diagnosing tuberculous lymphadenitis

Although IHC does not directly establish the diagnosis of tuberculosis, it may help exclude other conditions that produce granulomatous inflammation, such as T-cell lymphoma [n]. Special stains, including Ziehl–Neelsen, periodic acid–Schiff (PAS), and Grocott’s methenamine silver (GMS), may be used to detect mycobacteria or fungi within granulomas [n].

4.5. Application of IHC in diagnosing Lymphoma

IHC is a key tool for classifying lymphoma and distinguishing it from benign conditions [3].

• Differentiating Hodgkin and non-Hodgkin lymphoma:
  • Classical Hodgkin lymphoma: HRS cells are typically strongly positive for CD30 and CD15, negative for CD20, or weakly positive for PAX5 [3].
  • Non-Hodgkin lymphoma: IHC helps identify cell lineage (B cells or T cells) and classify subtypes based on characteristic markers. For example, diffuse large B-cell lymphoma is typically positive for CD20 and negative for CD3 [3]. T-cell lymphomas are generally positive for CD3 and other T-cell markers [3].
• Detecting micrometastases: IHC can identify small metastatic cancer deposits that may be overlooked on routine H&E staining by using markers specific to the primary tumor cells [n].

5. Comparison of histopathological and immunohistochemical features

6. When is Lymph node biopsy needed for diagnosis?

The decision to perform a lymph node biopsy is generally based on clinical assessment and the characteristics of the enlarged lymph node [n].

6.1. Findings that may indicate a need for biopsy

Lymph node biopsy may be indicated when:

• Lymphadenopathy persists for more than 2–4 weeks without a clear cause or does not respond to antibiotic or anti-inflammatory treatment [n].
• The enlarged lymph node is large, exceeding approximately 2–3 cm [n].
• The lymph node is hard, firm, immobile, or fixed to surrounding structures [n].
• Lymphadenopathy is accompanied by systemic symptoms such as unexplained weight loss, persistent fever, or night sweats (B symptoms) [n].
• The lymph node is located in an unusual or concerning site, such as the supraclavicular region [n].

6.2. Lymph node biopsy procedure

The lymph node biopsy process generally involves the following steps:

1. Initial assessment: Clinical examination, medical history review, and imaging studies such as ultrasound, computed tomography (CT), or magnetic resonance imaging (MRI) to determine the location and characteristics of the lymph node [n].
2. Biopsy: This may involve fine-needle aspiration (FNA), core needle biopsy, or excisional biopsy. Excisional biopsy is often preferred when lymphoma is suspected because it provides sufficient tissue to evaluate lymph node architecture and perform IHC [n].
3. Histopathological and IHC analysis: The biopsy specimen is sent to the anatomical pathology laboratory for processing, H&E staining, and any necessary IHC tests [1, 3].

7. The importance of accurate diagnosis and specialist consultation

7.1. Impact on treatment planning

Accurately identifying the cause of lymphadenopathy is essential because it determines the appropriate treatment plan.

• Reactive lymphadenitis: Usually requires treatment of the underlying cause of inflammation or clinical monitoring.
• Tuberculous lymphadenitis: Requires a prolonged, specific antituberculosis treatment regimen.
• Lymphoma: Requires a more complex treatment plan, which may include chemotherapy, radiotherapy, immunotherapy, or stem cell transplantation, depending on the lymphoma subtype and stage.

An incorrect diagnosis may delay treatment or lead to inappropriate management, potentially resulting in serious consequences for the patient.

7.2. The role of a multidisciplinary healthcare team

Accurate diagnosis and optimal treatment planning for enlarged lymph nodes require close collaboration among a multidisciplinary healthcare team, including clinicians in internal medicine, hematology, and oncology; pathologists; radiologists; and other specialists. Pathologists play a central role in examining tissue specimens and establishing a definitive diagnosis based on histopathology and IHC.

8. Frequently Asked Questions (FAQ)

1. Do enlarged lymph nodes always indicate cancer? No. Enlarged lymph nodes can result from many benign conditions, including infections and reactive lymphadenitis. However, if the enlargement persists, the lymph node is painless, hard, or firm, or systemic symptoms such as weight loss, fever, or night sweats occur, medical evaluation is necessary to exclude cancer [n].
2. Can blood tests distinguish between different causes of enlarged lymph nodes? Blood tests may provide useful clues, such as changes in white blood cell counts, erythrocyte sedimentation rate, or C-reactive protein (CRP). However, they are usually insufficient to establish a definitive diagnosis. Lymph node biopsy, histopathological examination, and IHC are often necessary for accurate differentiation [n].
3. Is lymph node biopsy painful? Lymph node biopsy is generally performed under local anesthesia or general anesthesia, depending on the procedure, so pain during the procedure is usually minimized. Mild discomfort may occur afterward and can be managed with pain medication [n].
4. What should I do if I notice an enlarged lymph node? If you notice an enlarged lymph node, particularly one that is painless, hard, or firm, or if it is accompanied by concerning symptoms, you should consult a physician for timely evaluation, diagnosis, and treatment recommendations [n].
5. What can immunohistochemistry detect that routine histopathology may not reveal? IHC can detect specific proteins on or within cells that may not be identifiable by morphology alone on H&E-stained sections. This helps determine cell lineage, assess differentiation, and identify important molecular characteristics for accurate disease classification, particularly in lymphoma [3].

9. References

• [1] Elmore, S. A. (2006). Histopathology of the Lymph Nodes. PMC. https://pmc.ncbi.nlm.nih.gov/articles/PMC1892634/.
• [2] van der Valk, P., & Meijer, C. J. (1987). The histology of reactive lymph nodes. PubMed. https://pubmed.ncbi.nlm.nih.gov/3499826/.
• [3] Cho, J. (2022). Basic immunohistochemistry for lymphoma diagnosis. PMC. https://pmc.ncbi.nlm.nih.gov/articles/PMC9057666/.

The information above is for reference purposes only and is not intended as medical advice. Please contact your doctor for detailed medical consultation.