Pathohistological diagnosis is a critical stage in the diagnostic and treatment process for every patient. It is performed by a pathologist who, based on various histological examinations, determines the presence or absence of a malignant neoplasm, its stage, degree of invasion, and the presence of metastases. These studies are facilitated by the pathohistological laboratory technician.
Immunohistochemical examination is essential for diagnosing many tumor diseases and is often decisive for determining the patient's treatment. This research involves a long and multi-stage process, requiring the laboratory technician to have detailed knowledge of each step. Fixation of the material, processing it into a paraffin block, antigenic recovery, and antibody incubation are all critical stages that can impact the success of the immunohistochemical reaction.
Knowledge of the antibodies and the antigens being targeted is crucial for selecting the appropriate protocol. Poor storage of antibodies or their improper dilution can lead to false-negative or false-positive results. During tissue processing for microscopic preparation, tissues are passed through a fixing solution, most commonly formalin, which "masks" many antigens. Thermal recovery is a necessary step for unmasking such antigens. Using an inappropriate antigen retrieval buffer can result in incorrect results. The incubation time of the antibody and the temperature at which the reaction occurs must align with both the antibody's sensitivity and specificity.
Visualization of the immunohistochemical reaction is most commonly performed using 3,3'-diaminobenzidine tetrachloride (DAB) chromogen, which produces a brown color. Counterstaining with hematoxylin, which stains nuclei blue, is recommended. The use of alcohol in this final stage can cause the loss of the reaction. Therefore, hematoxylin without alcohol in its composition, such as Mayer's hematoxylin, is preferred.
The purpose of this review is to examine the main stages involved in immunohistochemical studies, highlighting common errors and offering potential solutions for their prevention or correction.
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