Frozen section analysis | Histology

Frozen section analysis

This is necessary if the surgeon needs information about the tissue removed during an operation in order to decide on the course of the procedure. For example, a small malignant tumor is removed from the kidney. Now a quick incision is required to see whether the tumor has been completely removed or whether there is still massive amounts of malignant tissue at the edges of the tissue samples.

In the end, the outcome of the frozen section examination determines the course of the operation and the patient’s further therapy plan. How does a frozen section examination work? Within 10 minutes the tissue is stabilized by freezing at -20°C, then a 5 – 10 μm thick incision is made on the so-called microtome. This is placed on a slide, a small glass plate, and quickly stained. At the end, the findings are examined under the microscope and the result can be immediately forwarded to the operating room.

Staining methods

Many histological staining methods have developed over the last 120 years. The cell structures and tissues are divided into basophilic, acidophilic and neutrophil cells based on the color reaction with the staining agents. Furthermore, there are also agyrophilic and nucleophilic structures.

Basophilic stains everything that contains an acid group and is stained with a basic dye (for example hematoxylin or methylene blue). Structures that are acidophilic are basic and can therefore be stained with erosion or acid fuchsin (acid dyes). These include the cytoplasm and collagen fibers.

Neutrophilic or lipophilic components cannot react with either an acidic or a basic dye and therefore cannot be stained. Agyrophilic components can bind silver ions and convert them into elemental silver. A nucleophilic (nucleus = cell nucleus, cell nucleus-loving) color reaction results from nucleophilic dyes in the cell nucleus.These are DNA binding or basic substances that bind to nucleic acids.

Today, the well-tried chemical staining methods have been supplemented by immunological methods. The antigen-antibody reaction is used in this technique to detect certain cell properties. The reaction can then be made visible by a sophisticated technique.

Frequently used staining methods are:HE staining = Hematoxylin-Eosin staining: Hematoxylin, a natural dye, stains all structures blue, which are basophilic (= base-loving) and therefore acidic, such as DNA, cell nuclei, ribosomes, etc. Eosin, on the other hand, is produced synthetically. Eosin stains all cell structures red if they are acidophilic (= acid-loving) or basic.

The proteins of the cytoplasm, mitochondria, and collagen are among them. Azan staining: It is composed of the first letters of both colors, azocarmine G and aniline blue-gold orange: This stains the cell nucleus and muscle fibers red and the cytoplasm reddish. Collagen and reticular fibers turn blue in this staining.

The Giemsa stain (Giemsa’s Azure-Eosin-Methylene Blue) is used to stain blood cell smears. Cell nuclei can be easily recognized by the purple color reaction. The cytoplasm stains bluish.

In the elastica staining (resorcinol-fuchsin-orcein), all elastic fibers are shown in black-violet. The van Gieson staining method is characterized by the fact that staining is first performed with hematoxylin. Then picric acid fuchsin (micro fuchsin) or picric acid thiazine is used.

In the end, the cell nuclei appear black-dark brown, the cytoplasm appears rather light brown. Counterstaining with picric acid thiazine stains the elastic fibers and muscle tissue orange-yellow and the collagen fibers red. In trichrome staining according to Masson-Goldner, the size of the dye molecule is the most important factor in the staining method.

Iron hematoxiline is used, usually with three additional dyes, namely acid fox, orange G and light green. It stains collagenous connective tissue and mucus green, cell nuclei blue-black, cytoplasm red, muscles pale red and red blood cells (erythrocytes) orange-red. In addition, there is a Gram staining, which serves to differentiate bacteria.

Gram-positive bacteria are stained blue and Gram-negative bacteria are stained red. Ziehl-Neelsen staining is also used for bacteria, namely those that are acid-resistant and, for example, shows tuberculosis pathogens in red. Other staining methods that should be mentioned here are the Berlin-Blue reaction, which is responsible for the detection of trivalent iron ions in tissue sections, and the iron hematoxylin staining method according to Heidenhain.