Objective Assessment of Immunohistochemical Reactions

Abstract

Immunohistochemistry (IHC) has made a huge leap in development over the past 50 years. It has become a routine, and in a significant number of cases, a mandatory diagnostic method in medicine, and is also the most valuable and highly informative method of scientific research. Today, IHC often requires simply detecting a particular marker, which is insufficient; gradation of its expression also becomes necessary. The purpose of the work is to present methods for objectively determining the results of IHC reactions to assess their differences in groups of objects being compared. Materials - the search for primary sources of literature was performed using the information systems Scopus, Medline, PubMed Central, Google Secular. To assess the relative proportion of the cell array expressing a certain marker, we used the Crowe AR, Yue W. (2019) protocol with the freely available ImageJ software for deconvolution and downstream analysis. This allowed us to determine with a high level of reproducibility the specific level of membrane and/or cytoplasmic expression of a number of markers in regenerative neuromas in rats. However, the disadvantage of this method was the extremely limited ability to determine the intensity of expression of the markers under study. To compare the direct assessment of the intensity of expression of the IGH reaction in different objects, a method was proposed for determining the specific density of staining that occurs when performing the IGH reaction under the conditions of standardization of its parameters. To do this, we determine the optical density of selected objects on a digital image of a histological preparation using the Imag J program (Wayne Rasband NIH, USA). A list of conditions for conducting an IGH study has been determined, which standardize it and allow comparing the results in different groups. 1. The thickness of the sections should be standardized as much as possible. This is achieved by high-quality compaction of the material in paraffin and the use of a high-quality microtome. 2. The IGH reaction for subsequent comparison of results should be carried out with the same antibody (from one portion obtained). 3. The conditions for the IGH reaction should be standardized in terms of incubation time and temperature. We achieved this by simultaneously processing sections of several experimental groups by incubation with primary and secondary antibodies in a thermostat at 24°C (the temperature in the laboratory was 20-22°C) for 10-20 minutes, or at 4°C in a refrigerator for 16-24 hours. The second option showed better results. 4. Before performing the IGH reaction, it is advisable to titrate the primary antibody and determine its dilution, which will determine the optimal color intensity (first of all, it should not be excessive). 5. Microphotographs of preparations should be carried out under standardized lighting, exposure and resolution of the digital camera. Automatic photo mode is unacceptable. Conclusion: Immunohistochemistry and digital technologies have made a tremendous contribution to the development of medicine and biology. Their combination over the past 30 years has provided an unprecedentedly valuable tool for researchers. However, it requires compliance with a number of technological conditions that are necessary to obtain reliable and reproducible results.