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FISH protocol: Sample preparation

Can I use haematology probes on FFPE material and pathology probes on bone marrow samples?

Cytocell probes that begin with the catalogue classification LPH correspond to Haematology FISH probes and those that begin with LPS correspond to Pathology/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.

Pathology 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.

What is the recommended FFPE thickness to perform FISH?

Tissues specimens should be 3-4μm thick for optimal results. If sections are too thick, this can introduce difficulties in interpreting signals in different focal planes, distinguishing individual nuclei and poor hybridisation efficiency. If sections are too thin (<3 μm) this can lead to signal truncation.

How should tissue slides be stored prior to PETS FISH?

Only tissues preserved in 10% Neutral Buffered Formalin (NBF) and subsequently paraffin embedded or previously paraffin embedded sections are suitable for use.

NBF is most commonly used to preserve tissues and should be used for no longer than 3 months and then discarded. Extended formalin fixation time can cause major difficulties in PETS FISH such as reduced probe penetration, high levels of tissue auto-fluorescence, cell overlap or truncated nuclei and low hybridisation efficiency. Specimens should be fixed for between 18-24h in NBF, tissues fixed for longer will require Pepsin digestion times to be increased.

How does the use of pepsin help?

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 that the improvement is marked and 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.

What is the recommended mix when preparing fixative solution?

A solution of 3:1 Methanol/Acetic (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.

‘We would like to use peripheral blood smears; do you have a suggested method?’

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.

What are the optimum conditions for cell sample spotting?

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.

‘Our current in-house protocol recommends to bake and age the slides prior to probe application, your suggested protocol doesn’t mention this. Why is that?’

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.

When using the Cytocell FFPE Pre-treatment kit, how much ready to use enzyme should be applied to the tissue section?

When using the enzyme solution to digest your FFPE slide, liberally apply the enzyme ensuring the section is covered, as evaporation may cause the enzyme to recede from the edges and cause inconsistent digestion. When not in use, ensure that the enzyme solution is stored correctly at 2-8°C to preserve the enzyme activity.