CytoCell probes that begin with the catalogue classification LPH correspond to haematology FISH probes and those that begin with LPS correspond to solid tumour/haematopathology FISH probes.

The off-label use of probes is not recommended by CytoCell, the recommended protocol should be followed first to establish expected probe brightness and hybridisation efficiency before deviations from the protocol are considered. CytoCell can only guarantee results on the intended sample type.

Haematology probes have been optimised for use on 3:1 methanol/acetic acid fixed peripheral blood or 3:1 methanol/acetic acid fixed bone marrow.

Solid tumour probes have been validated for use on formalin-fixed paraffin-embedded (FFPE) tissue.

Haemato-pathology probes are validated for dual use on 3:1 methanol/acetic acid fixed bone marrow, 3:1 methanol/acetic acid fixed peripheral blood and FFPE tissue.

Pepsin treatment always improves FISH results, regardless of cell type used -peripheral blood, bone marrow, amniocytes, sputum, etc. CytoCell does not usually recommend pepsin treatment for every product because it is time consuming and requires additional costly reagents and for 'regular' cytogenetics preparations the benefits do not warrant the extra work.

We especially suggest that pepsin treatment be used for late gestational age samples because we have noticed a marked improvement certainly worth the additional effort. This does not mean that pre-treatment is essential or only required for late gestational samples; however, it can be beneficial as some people find it difficult to analyse FISH results without pre-treatment.

If you already routinely perform pepsin digestion for all samples then maintain this practice as it will produce the best results.

A solution of 3:1 Methanol/Acetic Acid (Carnoy’s) should be made freshly prepared, stored at -20°C and discarded after a day.

Try pre-fixing your bone marrow or peripheral blood samples by slowly adding ice-cold fix with agitation, immediately after the hypotonic treatment, to improve your cell preparations.

All labs should validate their own FISH protocol when working with peripheral blood smear preparations. As a suggestion, slides can be immersed in fixation solution from 10-30 mins, apply 10μl probe (or more if required, depending on the coverslip used), denature at 80°C for 5 mins* then follow the recommended CytoCell protocol.

*Please note that this has not been validated internally but based on user feedback.

The optimal conditions for spreading are: 25°C and 50% relative humidity (RH).

Recommendations:

• Ideally use a Thermotron^®, as it provides optimal conditions for sample spreading.
• Alternatively, suspend slide over a sink of hot water, or a 37°C water bath, then spot sample.
• Critical for all samples including peripheral blood.

In general, baking slides using a hotplate and/or ageing will produce weaker, more variable results. This tends to be part of older FISH protocols.