Immunohistological diagnostics are based on application of specific primary antibodies on cryo-fixed tissue section and following detection of coupled primary antibody by secondary antibody. Secondary antibody is conjugated with enzyme complex which could produce a precipitating coloured complex after use of staining solution.
For immuno-histochemical examinations sections are prepared of cryo-embedded biopsies by use of cryostat microtome. Therefore endomyocardial biopsy will be placed on pre-cooled (-20°C) metallic tray and covered completely by plastination glue Tissue-Tek. Tissue-Tek is freezing down immediately and preserve a hard consistence of embedded tissue.
Generally, cutting is performed for 3-5 slides for each antibody (about 20 cryo sections per patient) before immuno-staining is started. Then separated areas are processed with different antibodies accompanied with appropriate blocking and incubation steps and finally stained by an enzymatic conversion of dye AEC for producing red-colored immunospots for following microscopic examination. Second antibody and the colorimetric substrate are pre-mixed and well optimized for following digital image analysis. The final counterstaining of cryo sections is always carried out in staining machine (HE staining).
One microscopic slide is finally containing separated areas for 2 different antibodies. Hereby each following layer of cryo sections is placed in the field for the next antibody detection, i.e. in any area there are about 6 to 8 serial cryo sections. This cutting procedure ensure the more detailed analysis by simultane staining of different levels of biopsy by various antibodies.
Four sets of immunohistochemical staining are offered for specialized diagnostics of heart muscle tissue, whereas only the sets IC1-Heart muscle inflammation is proposed to perform in routine diagnostics procedure. Set IC2-Activation marker/Viral proteins is recommended in clinically suspected cases with high viral load of corresponding cardiotropic virus.
Coloured immunospots are counted digitally by application of in-house established digital imaging analysis software for calculating area fractions, numbers of immuno-spots and area of myocardial tissue (routine diagnostics). Values for counting are fixed by inclusion of digitally produced values in electronic database for in the final report. These report contains also numeric values for morphological characteristics like biopsy size, quality, fibrosis etc.
In general, immunohistochemical staining is performed on frozen sections of a second biopsy, not identical to histological examination. This procedure is benefical to reduce mis-interpretation by evaluation of only one endomyocardial biopsy.