Fluorescent In Situ Hybridization (FISH)
We currently offer diagnostic testing to detect invasive bacteria within fixed tissue samples, using Fluorescence in Situ Hybridization (FISH). Common indications for FISH include granulomatous or neutrophilic gastroenteropathies, unresponsive GI disease, inflammatory liver disease, unexplained granulomatous lesions of spleen, lymph node, or skin.
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Frequently asked questions:
- What is FISH?
- What are the indications for doing FISH?
- I have already taken biopsies, can I still submit samples for FISH?
- Can my regular histology lab do FISH?
- Why not just do a Gram stain?
- What are the limitations of FISH?
- Can you do FISH on any tissue? What about fluids and blood?
- Will the FISH result tell me which treatment to use?
- How much does it cost?
- What is the turnaround time?
1. What is FISH?
Fluorescent in situ hybridization, or 'FISH' is a technique used in molecular microbiology to identify bacteria within formalin fixed tissues. A fluorescent probe that binds to bacterial ribosomes in tissue sections can be visualized using a fluorescent microscope. Our analysis uses a screening 'all bacterial' probe, that binds to most bacterial species. If we identify bacteria, we can use a limited # of additional species specific probes to identify bacterial species (such as E. coli, Staph, Strep, Clostridia, Bartonella ). We do not currently offer probes to detect fungal, mycobacterial, or rickettsial organisms.
2. What are the indications for doing FISH?
FISH analysis of colonic biopsies is a vital adjunct to the diagnosis of Boxer dog colitis, in order to assess colon biopsies for evidence of intramucosal and intracellular bacterial invasion. If bacterial invasion is present, the invading species is usually E. coli, and we use a specific probe to detect E. coli infection. Diagnosis of bacterial invasion helps to formulate a treatment plan, in conjunction with colon culture and antimicrobial susceptibility data. It is important to appreciate that colon culture alone by no means confirmsE. coli invasion, and for maximal diagnostic yield, culture data must crucially be interpreted alongside FISH for spatial localization of bacteria.
FISH can also be very helpful in clinical cases where where bacterial involvement is suspected based on clinical or histological evidence, for example in chronic granulomatous diseases, cholangiohepatitis, endocarditis, suppurative pancreatitis, pyelonephritis, lymphadenitis, chronic cystitis.
3. I have already taken biopsies, can I still submit samples for FISH?
Yes. We do not require biopsies to be taken into special media for FISH analysis alone. We need a minimum of 5 unstained paraffinised sections, 4-5 microns thick, on adhesive coated or positively charged glass slides.
If, however, you have not already taken biopsies it may be helpful to contact us for advice. In some cases we may suggest that we send you a sampling kit to enable additional microbial analysis if warranted (e.g. 16S cloning, sequencing, microbial culture).
The exception is if you are investigating a suspected case of Boxer dog colitis. It is advisable to obtain a sampling kit from us prior to endoscopic biopsy, in order to culture the mucosa for E. coli antimicrobial susceptibilities. This is also very helpful for our ongoing research on Boxer dog colitis.
4. Can my regular histology lab do FISH?
It is extremely unlikely. But you can ask your usual pathologist to send recuts of slides direct to our lab for FISH testing. We must also receive payment (see 8 and 9) and a sample submission form before samples will be analyzed.
5. Why not just do a Gram stain?
The advantages of using FISH as opposed to Gram stain, is that there is much a higher likelihood of visualizing bacteria in tissues using fluorescent beacons. This is particularly true in highly cellular or inflamed tissues, where it can be very difficult to detect and differentiate bacteria from other cell types, inflammatory/necrotic debris and granules. FISH is a highly sensitive technique and can identify a single bacterium.
6. What are the limitations of FISH?
A negative FISH result does not completely exclude bacterial infection. Reasons for false negatives include the presence of dead/dying bacteria (they must be alive and metabolically active to take up the probe), low bacterial number, a patchy distribution of infection, overfixation, sulfasalazine treatment, and the presence of bacteria with thick cell walls (e.g. Listeria).
The eubacterial probe identifies most bacteria, and FISH MAY enable identification of the bacterial species present if we have the appropriate probe (though we have a limited # of species specific probes). It also does not provide any information on antimicrobial sensitivities.
False positives on FISH are also possible but unlikely, since we use additional probes as positive and negative controls.
7. Can you do FISH on any tissue?
Yes, we can perform FISH on any paraffin fixed tissue.
8. Will the FISH result tell me which treatment to use?
No, FISH does not give any information on antimicrobial susceptibility. But it may enable identification of the bacterial species present, and help to ensure an appropriate spectrum and penetration of antimicrobial cover (i.e. Gram + vs Gram -, intracellular vs extracellular).
9. How much does it cost?
For the first tissue: $190
For additional tissues (per tissue): $50
10. What is the turnaround time?
5-10 days, depending on how many probes we use. If formalin fixed tissue or paraffin embedded blocks are submitted, an additional 5-10 business days will be added to the turn-around time.