Chapter 10 Discussion Questions and Answers

10.1. With reference to chapter 8 compare and contrast the key steps of chromogenic in situ hybridization and immunocytochemistry.

Answer:

Immunocytochemistry	Step	In situ hybridization
Must be prompt and complete	Fixation	Must be prompt and complete
Heat induced or protease antigen retrieval	Sample pre-treatment	Proteolytic exposure of nucleic acids usually used
Application of protein solution in buffer	Blocking	Application of hybridization solution without probe
Simple application of unlabelled antibody in blocking buffer	‘Primary’ incubation	Complex hybridization solution, containing hapten-labelled probe, together with temperature of incubation used to control specificity of hybridization. May be followed with stringency washes.
Usually multistep to provide amplification	Detection system	Usually simple, often single enzyme-labelled antibody
Peroxidase most frequently used	Enzyme label	Alkaline phosphatase most frequently used
Short incubation	Substrate	Long incubation providing amplification

10.2 A series of FFPE sections from a head and neck cancer biopsy have been received to identify HPV status and subtypes present. How should the optimal proteinase K concentration be determined for the test sections? What internal quality controls should be run to ensure that the HPV probes are capable of detecting the virus?

Answer:

Optimisation of proteinase K concentration:

The first thing to consider is that the referred sections may not contain any demonstrable HPV at all! So, for the optimisation of the exposure of DNA using proteinase K a probe against a DNA sequence that will be present in all cells should be used. This could be a probe against a repetitive DNA sequence such as a chromosome specific centromeric alpha-satellite DNA.

The sections should be pre-treated with proteinase K using a minimum of two distinct conditions. This could be by applying the same concentration of the enzyme, but for different times or applying proteinase K at two concentrations for the same time (see Table 10.3 for suggested concentration range). In this way the results should either provide an optimal pre-treatment condition or indicate whether more or less severe proteolytic digestion is required. Please note that it is always important to examine the sections for gene demonstration and preservation of morphology. Ideally optimal DNA demonstration should be accompanied by excellent morphology, but if a compromise has to made then the emphasis should be placed on DNA signal intensity.

Internal quality controls to be run alongside the test samples should include:

A positive FFPE preparation for each probe or probe cocktail that is used. These could be sections of tissue samples or sections of blocks that contain cultured cells with different HPV sub-types. If referrals are frequent then the latter would be preferred as it is a renewable resource. It would also allow multi-block preparation and the possibility of mounting the resultant composite sections from this on the referred slides thus providing on slide validation of staining.

A negative control. This would be a section from the referred case. It would be taken through the technique, but either no HPV probe would be present in the hybridization solution or a scrambled DNA sequence ‘probe’ would be applied at this step.