Lymphoma is a cancer that originates in the lymphocytes, which are infection-fighting white blood cells that are part of the body’s lymphatic system. Diagnosis begins with histology, the study of tissue structure using a microscope. This foundational step allows pathologists to examine the physical appearance of abnormal cells and the overall architecture of the affected tissue, typically a lymph node. The visual evidence gathered is the initial basis for determining the specific type of lymphoma.
Preparing the Tissue Sample
The process of microscopic evaluation begins with obtaining a tissue specimen, usually through a biopsy of a lymph node. To preserve the cellular and architectural features, the sample is immediately placed in a chemical fixative, most often formalin. This fixation step prevents the tissue from degrading and prepares it for subsequent processing.
Following fixation, the tissue undergoes dehydration where water is removed using increasing concentrations of alcohol. This prepares the tissue to be infiltrated with melted paraffin wax, a hydrophobic substance that provides structural support. The tissue is then embedded in a block of this cooled wax, creating a formalin-fixed, paraffin-embedded (FFPE) block.
A specialized instrument called a microtome is used to slice the FFPE block into extremely thin sections, usually four to five micrometers thick. These slices are floated onto glass slides and stained to make the cells visible under the microscope. The standard technique is the Hematoxylin and Eosin (H&E) stain, which imparts the necessary color contrast for cellular analysis.
Hematoxylin is a basic dye that colors acidic components, such as the nucleus, a purplish-blue. Eosin is an acidic dye that stains the cytoplasm and extracellular matrix various shades of pink. This contrasting coloration allows the pathologist to clearly distinguish the nuclear features from the cellular body and the surrounding tissue environment, providing a general overview of the tissue’s structure.
Visual Clues Under the Microscope
The H&E-stained slide allows the pathologist to identify features suggesting that normal lymph node architecture has been replaced by malignant lymphocytes. Observations begin with the overall growth pattern of the abnormal cells. This pattern may be diffuse, meaning the entire tissue structure is overrun, or nodular, where the cells form distinct, rounded collections. The presence of a monotonous population of cells, rather than the varied mix seen in a healthy lymph node, strongly indicates a cancerous process.
Malignant lymphocytes often display significant structural abnormalities, referred to as atypia. The size and shape of these cancerous cells vary widely, from small cells similar to normal lymphocytes to very large cells with abundant cytoplasm. Pathologists closely examine the nucleus, which may exhibit irregular contours, deep indentations, or a clefted appearance, deviating from the typical smooth, round shape.
Within the nucleus, the chromatin (DNA and proteins) may be dispersed or clumped abnormally. A prominent nucleolus is frequently observed, suggesting high cellular metabolic activity and rapid division. Furthermore, an increased number of mitotic figures (actively dividing cells) indicates a high proliferation rate, a feature of aggressive cancers.
The surrounding microenvironment also provides important clues about the disease. In some lymphomas, malignant cells are intermixed with a varied inflammatory background, including reactive lymphocytes, plasma cells, and eosinophils. This inflammatory infiltrate is the body’s reaction to the tumor cells, and its pattern and composition contribute significantly to the initial diagnostic impression.
Categorizing Lymphoma by Cell Appearance
Microscopic evaluation on H&E slides provides the initial separation into the two major categories: Hodgkin Lymphoma (HL) and Non-Hodgkin Lymphoma (NHL). The distinction is based on the presence or absence of the highly specific malignant cell known as the Reed-Sternberg cell. Finding this cell is the defining criterion for diagnosing Hodgkin Lymphoma.
The Reed-Sternberg cell is a large, often multinucleated cell characterized by its distinctive appearance, frequently described as resembling “owl’s eyes.” This is due to its bilobed nucleus and large, prominent nucleoli surrounded by a clear halo. Although the hallmark of HL, these malignant cells constitute only a small percentage of the total biopsy sample, which is mostly composed of reactive lymphocytes and inflammatory cells recruited by the Reed-Sternberg cells.
Non-Hodgkin Lymphoma is diagnosed when the Reed-Sternberg cell is absent and the proliferation consists of a monomorphic population of abnormal B-cells or T-cells. NHL subtypes are broadly categorized based on the size and maturity of the proliferating lymphocytes. Low-grade lymphomas typically involve small, mature-appearing cells that divide slowly and often display a nodular growth pattern.
High-grade lymphomas are characterized by a diffuse growth pattern and the proliferation of large, highly atypical cells with rapidly dividing features. For example, malignant cells in Diffuse Large B-cell Lymphoma have large nuclei, abundant cytoplasm, and numerous mitotic figures. This initial morphological classification provides a framework to narrow down the specific subtype, which is crucial for determining treatment.
Using Advanced Markers for Confirmation
While basic H&E histology provides the initial classification, advanced techniques are necessary to definitively identify the lineage and specific subtype of lymphoma. Immunohistochemistry (IHC) uses antibodies to detect specific protein markers expressed on or inside the malignant cells. The antibodies are tagged with a visible chromogen, allowing the pathologist to visualize which cells express a particular marker.
For example, B-cell lymphomas typically express the CD20 marker, while T-cell lymphomas express CD3. Markers like CD30 and CD15 are used to confirm Classical Hodgkin Lymphoma, as they are expressed by the Reed-Sternberg cells. This method is useful because it maintains the tissue architecture, showing the exact location of marker-expressing cells within the microenvironment.
Flow cytometry is another powerful technique that analyzes the physical and chemical characteristics of thousands of individual cells in suspension. This method uses fluorescently labeled antibodies to simultaneously assess multiple cell surface and intracellular markers. Flow cytometry offers high sensitivity, allowing for the detection of subtle differences in antigen density that distinguish between various lymphoma subtypes.
These advanced studies confirm the cell of origin (B-cell or T-cell) and identify specific markers that determine the precise subtype. The combination of H&E morphology and the immunophenotype profile from IHC and flow cytometry provides the comprehensive diagnosis required for effective treatment planning. This integration ensures the diagnosis is based on a detailed molecular signature, not solely on visual appearance.