Diffuse Large B-cell Lymphoma (DLBCL) is a common and aggressive form of non-Hodgkin lymphoma, a cancer originating in the lymphatic system’s B-cells. Its diagnosis is dependent on the microscopic analysis of tissue, a practice known as histopathology. By examining a tissue sample from a lymph node, pathologists can identify the defining characteristics of the disease, making an understanding of these visual markers a primary step in diagnosis.
Understanding Histological Examination in DLBCL
Histology is the microscopic study of tissue anatomy. The procedure begins with a biopsy, where a small piece of tissue, often an entire lymph node, is surgically removed. This tissue is then preserved through fixation, usually in a formalin solution, which prevents decay and maintains the cellular structure. Following fixation, the tissue is processed, embedded in a block of paraffin wax, and cut into extremely thin slices.
These translucent sections are mounted onto glass slides for staining. The most common staining method used is Hematoxylin and Eosin (H&E). This technique provides color contrast, staining cell nuclei a purplish-blue and the cytoplasm pink. A pathologist, a physician specializing in disease diagnosis through tissue examination, then analyzes these prepared slides to assess the tissue’s architecture and individual cell characteristics.
Core Microscopic Characteristics of DLBCL
When a pathologist examines a suspected DLBCL sample stained with H&E, they look for specific hallmark features. The first is a “diffuse” growth pattern. In a healthy lymph node, immune cells are organized into neat structures called follicles; in DLBCL, this architecture is effaced and replaced by dense, disorganized sheets of cancerous lymphocytes.
The second feature relates to the cancer cells themselves, which are characteristically “large.” These malignant B-cells are noticeably bigger than normal, resting lymphocytes. A pathologist often uses the nucleus of a resident macrophage as an internal ruler; the cancerous B-cells in DLBCL have nuclei that are the same size as or larger than a macrophage nucleus.
The nuclei of the large B-cells are often described as vesicular, meaning the chromatin is fine and pale-staining. These nuclei frequently have irregular shapes and contain one or more prominent nucleoli. The cytoplasm is variable but often appears as a thin, blue-staining rim. A high number of mitotic figures, which are cells caught in the act of dividing, are also seen, reflecting the cancer’s rapid growth.
Common Morphological Variants within DLBCL
While all DLBCL cases share the core features of a diffuse growth of large B-cells, pathologists recognize several morphological variants based on the predominant cell appearance. The most common variant is the centroblastic type. This form is dominated by cells called centroblasts, which are large cells with round or oval nuclei, fine chromatin, and typically two to four distinct nucleoli that are often located near the nuclear membrane.
Another recognized variant is the immunoblastic type. This variant is characterized by the presence of immunoblasts, which are also large cells but differ from centroblasts. Immunoblasts have a single, prominent nucleolus located in the center of the nucleus and generally possess a greater amount of basophilic cytoplasm, giving them a deeper blue appearance.
A less common but important variant is the anaplastic type. The cells in this subtype are very large and show marked pleomorphism, meaning they have highly irregular shapes and sizes. These cells can sometimes be mistaken for the cells seen in other cancers, such as Hodgkin lymphoma or even non-lymphoid cancers like carcinomas.
The Role of Special Stains in DLBCL Diagnosis
H&E staining reveals the architecture and cell morphology, but it cannot definitively prove the cancerous cells are B-cells. To confirm the cell of origin and gather more information, pathologists employ immunohistochemistry (IHC). This method uses manufactured antibodies designed to bind to specific proteins, or antigens, present on or inside the cells. When the antibody binds to its target antigen, a chemical reaction produces a visible color, effectively tagging cells that express the protein.
For DLBCL, a panel of IHC stains is applied to the same tissue sections. To confirm a B-cell lineage, stains for proteins like CD20, CD79a, and PAX5 are used. CD20 is a robust marker found on the surface of most B-cells, and its strong, uniform presence across the large, abnormal cells is a classic finding in DLBCL.
Beyond confirming lineage, IHC helps assess the tumor’s behavior. The Ki-67 stain is used to measure proliferation. This marker detects a protein present only in actively dividing cells, and in DLBCL, the Ki-67 index is usually high, often above 40%. Other IHC markers, such as BCL2, BCL6, and MYC, can also be stained to provide information that helps classify the lymphoma and can have prognostic implications.
Distinguishing DLBCL from Other Lymphoid Malignancies Histologically
A pathologist’s task includes differentiating DLBCL from other cancers that can appear similar under the microscope. For instance, Hodgkin lymphoma can also present with large, abnormal cells, but these are classic Reed-Sternberg cells, which have a different appearance and are set in a background of mixed inflammatory cells. The IHC pattern is also distinct, as Hodgkin lymphoma cells are typically positive for CD30 and CD15 but negative for the B-cell marker CD20.
Another condition to distinguish is Burkitt lymphoma, another aggressive B-cell lymphoma. While its cells are also rapidly dividing, they are more uniform in size and shape than those in DLBCL. Burkitt lymphoma is known for its extremely high proliferation rate, with a Ki-67 index approaching 100%, and often displays a “starry sky” pattern, where scattered benign macrophages are seen among the cancer cells.
Other lymphomas, such as anaplastic large cell lymphoma (ALCL), can mimic the large-cell appearance of DLBCL. However, ALCL is usually a T-cell lymphoma, a fact confirmed when IHC stains are negative for B-cell markers like CD20 but positive for T-cell markers like CD3. Certain high-grade B-cell lymphomas share morphological features with DLBCL but are defined by specific genetic rearrangements.