FAQ's Troubleshooting

How do I place an order if an authorized distributor is not within my region?

If no authorized distributor is located within your region please contact us at purchase@cancergeneticsitalia.com to place an order.

How do I become a distributor of CGI Italia’s products?

To become a distributor of our products, please contact us at contact@cancergeneticsitalia.com.

What are the storage and shipping conditions for CGI Italia’s products?

Our products are stored ‐20oC and are shipped on blue ice. They can be shipped on dry ice upon specific request from the recipient. Please note that certain courier do not accept to ship dry ice. Additional fee will apply.

How are your DNA-FISH products shipped (Courier or Post)?

Our DNA-FISH products are shipped via courier services, such as UPS, FedEx, or DHL.

What’s the packing size of your probes? Do you provide your products as a FISH kit?

Our probes are provided in a ready‐to‐use 100 μL format (10 tests/vial), which reduces reagent waste and preparation time. The probes are retailed individually.

Are your products compliant with regulatory requirements?

Yes, our products are CE marked per EU regulatory requirements for safety and effectiveness.

What specimen types are applicable to your products?

Our probes can be used on bone marrow, peripheral blood, or formalin‐fixed paraffin‐embedded (FFPE) specimen types.

What is the advantages and limitation of fluorescence in situ hybridization (FISH)?

The primary advantage of the FISH technique is its applicability to non‐dividing cells and a variety of specimen types. FISH is a preferred method for diagnosis, prognosis, treatment response, and minimal residual disease detection in a variety of hematopoietic neoplasms and solid tumors. The technique is able to detect various chromosomal abnormalities, such as gains, losses, and translocations.

The limitations of FISH include the inability to detect certain micro‐deletions due to the resolution limit of FISH. FISH is considered an adjunct to other technologies including conventional cytogenetics. A medical decision cannot be made based on the result of a FISH assay alone.

What’s the time requirement for completing a FISH assay?

The average time for completing a FISH assay is ~24hrs when using a Probe intended for hematologic malignancy , and ~48hrs when using a Probe intended for solid tumor.

Why do I observe weak signals and how can I correct this?

Weak signals may be observed due to:

  • Use of an old mercury lamp (run time >200 hours); replace the mercury lamp after 200 hours of run time.
  • Use of unsuitable immersion oil; use immersion oil with no auto-fluorescence in the UV range.
  • Use of unsuitable filters; use the appropriate filter sets. Also, check that the excitation and emission of the dyes fall within the range claimed by the filter manufacturer. The filter specifications table identifies the filter requirements for Cancer Genetics Italia DNA-FISH Probes. Also, be sure the filters are well maintained and have no visible damage.
  • Inadequate denaturation of the DNA-FISH Probe; increase the denaturation temperature and/or duration. Optionally, the specimen may be treated with pepsin prior to denaturation.
  • Insufficient pretreatment of the specimen (visible cytoplasm on the specimen appearing as a green or yellow haze). In such cases it may be beneficial to perform the optional pretreatment steps outlined in the Instructions for Use (IFU).
  • Baking or aging the slide for too long; check the temperature of the hot plate or oven that is used to age the slide.
  • The time and temperature routinely used to artificially age the slide varies among specimen types and laboratories. Adjust conditions according to laboratory SOPs.

Why I see areas without signals?

There may be areas without signals due to:

  • Presence of air bubbles during hybridization, or application of an insufficient volume of Probe.
  • Before adding the cov¬erslip, gently remove all air bubbles with a pipette tip or needle. After adding the coverslip, gently remove any air bubbles by rolling a pencil eraser evenly across the coverslip. Also, ensure there is sufficient Probe volume (10 μl Probe/22×22mm target area). Increase or decrease the volume of Probe and size of coverslip proportionally with any increase or decrease in the size of the target area.
  • Low permeability of the specimen to the Probe, which results in resistance to hybridization. Optionally, the specimen may be treated with pepsin prior to denaturation (protocol is provided in the IFU).

Why are the signals fading?

The signals may be fading due to:

  • Photobleaching of the slide; always close the microscope shutter when not observing the specimen. It is important that hybridized slides are stored at 4°C and protected from light. Fluorescently labeled probes are readily photobleached by exposure to light, both before and after hybridization. To minimize photobleaching, handle Probes and hybridized specimens in reduced light as much as possible.
  • Use of immersion oil with auto-fluorescence in the UV range. Be sure to use immersion oil with no auto-fluorescence in the UV range.
  • Use of expired or oxidized Antifade/DAPI, which will appear deep purple/brown in color. Verify the expiration date of the Antifade used.
  • Image acquisition. Some photobleaching will occur from extended exposure to the UV source during image acquistion.

Why do I see no signals?

There may be no signals due to:

  • Application of an insufficient Probe volume and/or lack of cells in target area. It is good practice to check the slide under a phase contrast microscope, prior to hybridization, to select appropriate areas for hybridization.
  • Inappropriate denaturing/incubation conditions. Check the temperature of the controlled hot plate prior to use and ensure that the hot plate is calibrated regularly. Make sure to respect the recommended time and temperature for denaturation/incubation, and ensure that the incubation chamber is humidified, as recommended in the IFU (provided with the product and available at www.cancergeneticsitalia.com).
  • Too much cytoplasm present (appears as a green or yellow haze), which impedes the Probe from hybridizing to the target DNA. Optionally, the specimen may be treated with pepsin prior to denaturation (protocol is provided in the IFU).
  • Specimen quality not optimal for FISH. Occasionally a specimen will not lend itself to be processed for FISH. This may be the case in a subset of formalin-fixed paraffin embedded (FFPE) specimens when a variety of involved tissues (such as bone) may inhibit processing of the slide, and yield poor or no FISH signals.

Why is the nuclear morphology compromised?

The nuclear morphology may be compromised due to:

  • Over-denaturation of the specimen; reduce the duration and/or temperature of denaturation. It is important tocheck the temperature of the hot plate or oven used to age the slide.
  • Excessive pretreatment of the specimen, which leads to chromatin loss; reduce the pepsin concentration and/or incubation time during pretreatment, or eliminate this optional step altogether.

Why is there a high level of background noise?

A high level of background noise may be due to:

  • Use of an unclean slide during specimen preparation; clean slide with 70% ethanol and wipe with a lint free cloth prior to dropping the specimen on the slide.
  • Presence of cellular debris in the specimen; wash the specimen pellet in fresh fixative prior to dropping. Allow residual debris to settle to bottom of tube before drawing up the specimen.
  • Allowing the slide to dry during either hybridization or post-hybridization washes; when placing coverslip over target area, ensure proper sealing with rubber cement to prevent the Probe from drying. Also, do not allow slide to dry after removing coverslip for post-hybridization washes, or in between the washes.
  • Inadequate post-hybridization washing; ensure the wash buffers have reached the optimal temperature recommended in the IFU prior to starting the washes. Also be sure to wash the slide for the duration recommended in the IFU.

Why is there a high level of nuclear background noise?

A high level of nuclear background noise may be due to:

  • Inadequate post-hybridization washing; although rarely observed, a high level of nuclear background noise may be reduced by increasing the washing stringency and/or duration.