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3.1 What are the similarities and distinctions between the techniques of immunocytochemistry, flow cytometry and in situ hybridisation?
All three technologies can be applied to intact cells; immunocytochemistry and in situ hybridisation can additionally be used on tissue sections. ICC and flow cytometry employ antibodies that bind to antigens to identify proteins. In contrast ISH uses nucleic acid probes that identify DNA and RNA sequences. ICC read outs are semi-quantitative. Those for ISH may be semi-quantitative or quantitative, while that for FC is quantitative.
3.2 What factors will affect the specificity of a primary antibody?
The main factor affecting primary antibody specificity is epitope recognition. When the epitope is shared across more than one protein then cross reactivity can occur. A secondary factor is whether a polyclonal or monoclonal antibody is used. The former will contain a ‘soup’ of antibodies each of which will be reactive with distinct epitopes. By contrast a monoclonal antibody interacts with a single epitope only and accordingly the risk of cross reactivity is lower, but cannot be excluded.
3.3 What options are available for epitope retrieval for preparations that have been formalin fixed?
There are two main options; protease induced epitope retrieval (PIER) and heat induced epitope retrieval (HIER). The former needs to be carefully controlled to avoid under-digestion resulting in suboptimal exposure of an antigen and over-digestion that can liberate antigen from the preparation and destroy cell/tissue morphology. PIER may be ineffective in revealing antigens in tissue preparations when formalin fixation exceeds several days. In contrast fixation time usually has less influence on antigen exposure or sample preservation when HIER is used.
3.4 What are the key functional components of a flow cytometer?
A flow cytometer consists of a fluidic system which delivers the fluorescently labelled cells in suspension to the laser beam in a thin stream, coupled with an optics-to-electronics system to record the light scattering and fluorescence emission on a cell-by-cell basis and compute the data.
3.5 What is the main technical challenge encountered when undertaking flow cytometry of whole blood?
The main technical challenge derives from the preponderance of red blood cells over white blood cells and the potential introduction of artefacts during the process of sample separation.
3.6 What affects fluorescence intensity and light scattering?
Fluorescence intensity is affected by the number of antigens on the cell surface and within the cells, while light scattering is influenced by the size and granularity of the cells.
3.7 How does the physical separation of cellular sub-populations happen?
The physical separation of cells results from the disruption the sample stream caused by a vibration mechanism that creates thousands of individual droplets. Some of the droplets contain one cell and according to the characteristics of the contained cell the droplet will be electrically charged and differentially deflected for collection.
3.8 What are the principal chemical components and physical factors will affect the formation of base pairing between a nucleic acid probe and a target gene sequence in an ISH procedure?
Primary chemical components include monovalent (salt) concentration and inclusion of formamide. The greater the concentration of the former the less repulsion there will be between the nucleic acid sequences thereby driving hybrid formation. Formamide acts as a helix destabilising reagent and allows specific hybrid formation to occur at near physiological temperatures with the consequent conservation of cell and tissue integrity. The temperature used during hybridisation will affect hybrid formation. Higher temperatures will favour specific complementary base pairing. Longer hybridisation times will also favour hybrid formation. These chemical and physical factors need to be carefully controlled to ensure that specific hybridisation is achieved.