21 Molecular pathology
Introduction
All of this leads to the role the histology laboratory plays in molecular pathology. In the histology laboratory the main method used in molecular pathology is ISH. John et al. (1969) and Gall and Pardue (1969) described the technique of ISH almost simultaneously.
• DNA and mRNA are not as sensitive to formalin fixatives.
• Probe-target hybrid is stronger than antibody-antigen complex.
• Provides an alternative means of detection when reliable antibodies are not available.
Applications
There are many modifications of ISH methods that relate to application needs. Although the demonstration of DNA and RNA sequences by ISH is a valuable research tool, according to Warford and Lauder (1991) and Mitchell et al. (1992), it is also used diagnostically in:
ISH can provide cytological information on the location and alteration of genomic sequences in chromosomes. Traditionally, the technique has been applied to metaphase chromosome spreads (Davis et al. 1984; Lux et al. 1990), but it has been shown to be applicable to interphase nuclei (Hopman et al. 1988; Poddighe et al. 1992). Routine paraffin wax preparations of tissues can be used and ‘interphase cytogenetics’, as the method is termed, can provide direct information on chromosomal abnormalities in unselected tumor cell populations.
Viral identification can be undertaken using a variety of methods, of which only immunohistochemistry and ISH provide simultaneous morphological information. The sensitivity of immunohistochemistry for the visualization of viral antigens, and ISH for the demonstration of cytomegalovirus, correlate well (Van den Berg et al. 1989). Most viral ISH methods use probes for DNA. Others, such as in the demonstration of the Epstein-Barr virus (Fig. 21.2), the detection of a virally encoded RNA transcript, provide results that are more sensitive than the use of antibodies and may even approach that of the PCR (Pringle et al. 1992).

Figure 21.2 Example of automatic chromogenic in situ hybridization (CISH) staining.
(a) Epstein-Barr virus-encoded RNA (EBER) and (b) cytomegalovirus (CMV).
(Photographs courtesy of Leica Microsystems, Inc.)

Figure 21.3 Example of automatic chromogenic in situ hybridization (CISH) staining (a) kappa (b) lambda.
(Photographs courtesy of Leica Microsystems, Inc.)
Chromogenic in situ hybridization (CISH) is a method ‘that enables the detection of gene expression in the nucleus using a conventional histochemical reaction’ (White 2005); it is used for the detection of abnormal genes and to identify a gene therapy treatment direction. CISH can be used as an alternative in screening archived breast cancer tissue samples for HER2/neu (type 1 growth factor receptor gene) (Madrid & Lo 2004). Automated CISH techniques were used for detecting light chain expression in paraffin sections on plasma cell dyscrasias and B-cell non-Hodgkin lymphomas ‘appeared superior to IHC’ in that the ISH resulted with no background staining (Beck et al. 2003).

Figure 21.4 Example of automatic silver (silver deposition technology) in situ hybridization (SISH) staining.
(a) HER2 and Chr17. (b) HER2 and Chr17.
(Photographs courtesy of Ventana Medical Systems, Inc.)
Polymerase chain reaction-ISH (PISH) is another form of ISH. Viral RNA is detected by RT-PCR, using formalin-fixed paraffin-embedded tissue (FFPE). PISH results have been compared to IHC on staining for Newcastle disease in veterinary medicine. Newcastle disease is an avian viral infection that has a potential for rapid spread and may cause serious economic impact and international trade restrictions in the poultry industry (Wakamatsu et al. 2005). PISH is also used in the detection of human papillomavirus in uterine cervical neoplasia (Xiao et al. 2001).
Another area in which in situ hybridization and immunochemistry can be viewed as complementary techniques is in the phenotyping of tumors. Many monoclonal and polyclonal antibodies are available for phenotyping and these may be employed in sensitive and rapid techniques. When problems arise in the interpretation of immunohistochemical results, mRNA phenotyping by in situ hybridization can be helpful (Pringle et al. 1990, 1993; Kendall et al. 1991; Ruprai et al. 1991).
Common reagents
1. Diethylpyrocarbonate (DEPC) treated water
2. 2% aminoalkylsilane (positively charged slides)
These slides may be purchased pre-coated
Make sure they are RNA/DNA free
Aliquot and freeze below −20°C
5. 0.1 M triethanolamine (TEA), freshly made
6. 1 M Tris (this is to make buffers that vary in pH: buffer #1, pH 7.5; buffer #2, pH 9.5)
9. Maleic acid buffer a washing buffer
10. 20× Saline sodium citrate (SSC) buffer (this is also used to make 2× SCC and 1× SCC buffers)
(may cause increase in background)
14. Detection method reagents:
15. Colorimetric detection reagents:
Probes and their choice
Probe preparation and labeling
• Direct: the reporter molecules (enzyme, radioisotope or fluorescent marker) are directly attached to the DNA or RNA.
• Indirect: a hapten (biotin, digoxigenin, or fluorescein) is attached to the probe and detected by a labeled binding protein (typically an antibody).
Methods for incorporating labels into DNA are nick translation and random primer methods.
Preparation of the dilution series
1. Dilute the labeling probe using dilution buffer to a starting concentration of 2.5 pmol/µl.
2. Make a dilution series of purified probe in Eppendorf tubes to give nucleic acid concentrations of 300 pg/µl, 100 pg/µl, 30 pg/µl, 10 pg/µl, 3 pg/µl, and one tube containing diluent only. Ensure that all tube volumes are equal. Repeat the same dilution series with your control or used pre-labeled (with control) test strips.
3. Apply 1 µl drops from each tube onto the nylon membrane (Roche). The control dilutions should be lined up with the test sample dilution concentration. For an example of placing spots, see Figure 21.5.
4. Label the position of each application with a pencil on the side of the strip (not on the strip).
5. Fix the nucleic acid to the membrane by either baking the membrane for 30 minutes at 120°C or using a UV light.
6. Wash the membrane briefly in washing buffer.
7. Immerse in blocking solution for 10 minutes.
8. Incubate with reagents used in ISH detection technique.
Note: Dilute reagents in blocking solution and use this solution for washing.
9. Detect enzyme using the same solutions and procedure as for ISH method.