Application of molecular probes in histopathology of communicable diseases

The use of molecular probes in histopathology is based on the hybridization of labelled DNA and RNA probes with nucleic acid sequences in samples of diseased tissues. The main techniques are (1) in situ hybridization (ISH), where labelled probes are used to detect complementary DNA or RNA in histological sections, and (2) examination by Southern or other blotting techniques of nucleic acids extracted from homogenized biopsy material. Of these ISH enjoys better rapport with histopathologists because of its similarity to immunohistochemistry. It has the unique advantage over other molecular biology techniques of allowing localisation and visualisation of target nucleic acid sequences within morphologically identifiable cells or cellular structures. Probes for ISH may bear radioactive or non-radioactive labels. Isotopic probes (3H, 32P, 35S, 14C, 125I) are generally more sensitive than non-isotopic ones, although they are less stable and require longer processing times. Numerous non-isotopic labels have been explored; of these biotin and digoxigenin are the reporters of choice. In ISH, finding the optimal balance between good morphological preservation of cells (which is dependent on tissue fixation) and strong hybridization signals, is crucial. Tissues fixation and retention of cytoskeletal structures, unfortunately, impede diffusion of probes into tissues. ISH sensitivity is also influenced by inherent properties of the probe and hybridization conditions. Although ISH is largely a research tool in histopathology, it is already making strong inroads into diagnostic histopathology. It has been applied for the detection of CMV, HPV, HIV, JC virus, B19 parvovirus, HSV-1, EBV, HBV, hepatitis delta virus, Chlamydia trachomatis, salmonella and mycoplasma etc. Its application includes localisation of sites of infection, elucidation of mechanisms of virus transmission and dissemination and investigation of the link between virus infections and cancer. Demonstration of mRNA transcripts in cells by ISH is a useful adjunct in elucidating the pathophysiology of disease. (author). 15 refs, 1 tab