When a tissue sample is taken for a biopsy, it is sent to a pathology lab for examination. The first step in this process is often Hematoxylin and Eosin (H&E) staining, a technique used for over a century. This method gives pathologists a detailed view of the tissue’s microscopic structure, or histology. By coloring different parts of cells in contrasting shades, H&E staining reveals the size, shape, and arrangement of cells, providing the visual information needed to determine if cancer is present.
The Science of H&E Staining
Before viewing under a microscope, a tissue sample is prepared. First is fixation, where the tissue is preserved in a formaldehyde solution to prevent decay and stabilize its structures. The sample is then processed to remove water, which is replaced with paraffin wax. This embeds the tissue into a solid block, making it firm enough to be cut into thin slices.
Using a microtome, the paraffin-embedded tissue is sectioned into slices a few micrometers thick and mounted onto a glass slide. The colorless tissue requires staining to make its components visible. The slide is first submerged in hematoxylin, a basic dye that binds to acidic components. This gives the cell nucleus, which is rich in nucleic acids like DNA, a distinct blue or purple color.
After applying hematoxylin, the slide is treated with eosin, an acidic dye attracted to basic elements in the tissue. Eosin stains the cytoplasm, connective tissue, and other proteins in shades of pink and red. This two-part coloring process provides a clear contrast between the nucleus and the rest of the cell, revealing detailed structural information.
Visualizing Cancerous Tissue
Pathologists examine H&E-stained slides for morphological changes that distinguish cancerous from healthy tissue. Normal cells are uniform in size and shape and arranged in an orderly pattern. Cancerous tissue, in contrast, often loses this architecture and appears disorganized. The cells may grow in sheets or clusters that invade surrounding structures, disrupting natural boundaries.
A focus is on the appearance of the cell nucleus, stained blue by hematoxylin. In cancer cells, the nuclei often exhibit abnormalities. They may be larger and more varied in shape than normal cells, a feature known as pleomorphism. The nuclei can also appear unusually dark, a condition called hyperchromasia, resulting from an increased amount of DNA. These alterations reflect the genetic instability that drives malignant growth.
Another feature is the nucleus-to-cytoplasm ratio. In a healthy cell, the nucleus occupies a proportional amount of the cell volume. Cancer cells frequently have a much higher ratio, meaning the nucleus is disproportionately large compared to the cytoplasm. This change indicates malignancy, as it points to a cell focused on replication and division over its normal functions.
Pathologists also look for evidence of increased cell division, or mitotic activity. Cell division is tightly controlled in healthy tissues, but in cancerous tissue, this control is lost, leading to unregulated proliferation. Under the microscope, this appears as an increased number of mitotic figures, which are cells captured in the phases of division. Numerous or abnormal mitotic figures signal aggressive growth.
The Role of H&E in Cancer Grading
Beyond identifying cancer, H&E staining is used to determine a cancer’s grade. Grading is a system for classifying a tumor based on how abnormal its cells and tissue structure appear. This measures cellular differentiation—how much the cancer cells resemble the healthy cells of the original tissue. The features observed in the H&E stain, like nuclear irregularity and architectural disorganization, are used in this process.
A well-differentiated, or low-grade, cancer is composed of cells that look and are organized much like normal cells. These tumors tend to grow and spread more slowly and are associated with a better prognosis. For example, a Grade 1 tumor has cells that are only slightly altered from their normal counterparts, retaining much of their original structure.
Conversely, a poorly-differentiated or undifferentiated cancer is considered high-grade. The cells in these tumors look abnormal and bear little resemblance to healthy cells, often appearing disorganized and primitive. High-grade cancers, such as Grade 3 or 4, are more aggressive, meaning they are likely to grow and metastasize quickly. This grade helps oncologists predict the cancer’s behavior and develop a treatment strategy.
Complementary Diagnostic Techniques
H&E staining provides a map of tissue morphology but is often the first step in diagnosis. It reveals cell appearance and arrangement but cannot identify the specific molecules they produce. When a diagnosis is uncertain or targeted therapies are considered, pathologists use complementary testing methods for a more complete understanding.
One common follow-up test is immunohistochemistry (IHC), which uses antibodies to detect specific proteins in or on cancer cells. For instance, IHC can identify hormone receptors in breast cancer or markers that reveal a tumor’s origin when it has spread. This information, not visible with H&E staining alone, guides treatment decisions, like whether a patient might respond to hormone therapy or other targeted drugs.
In some cases, a deeper genetic analysis is required. Molecular tests like fluorescence in situ hybridization (FISH) can visualize and count copies of specific genes within the nucleus. This is useful for detecting gene amplifications, like the overexpression of the HER2 gene in some breast and stomach cancers, which has direct implications for treatment. These advanced techniques build upon H&E findings, adding molecular and genetic information to create a precise diagnosis.