FAQ on ImmunoCyt™ / uCyt+™
This section answers frequently asked questions by professionals with regard to the use of ImmunoCyt™ / uCyt+™. Questions are classified under the following five topics:
Urine specimen collection
Could bladder wash (by cystoscopy or catheter) be used for the test?
No. The urine sample should be collected from voided urine and must not come from first morning urine.
Can the test be performed with urine from bladder reconstruction using intestine segments?
No. This could create a false positive result, in particular with the green fluorescent signal.
What is the required volume of urine for the test?
A minimum of 20 ml of urine.
What kind of alcohol can be used at the collection center?
Ethanol or isopropyl alcohol 50%. Denatured alcohol and methanol cannot be used.
What kind of container should be used?
Transparent leak proof plastic container with 80 ml – 90 ml graduation.
What volume of ethanol or isopropyl alcohol 50% should be added to the urine specimen?
Equal volume with urine sample.
When should the ethanol or isopropyl alcohol 50% be added to the urine sample?
Within one hour from time of collection.
When should the urine specimen be received by the laboratory?
Within five days from collection date (kept in refrigerator), since it has to be treated with ImmunoCyt™/uCyt+™ within seven days from collection date.
With the Thinprep® instrument, can urine samples be collected in CytoLyt® solution?
Please refer to the section “Procedure” below.
Reagents
What is the composition of the ImmunoCyt™/uCyt+™ fixative?
The ImmunoCyt™/uCyt+™ fixative is made of active ingredients (such as buffer and polyethylene glycol) to better preserve cell structures and their antigenic sites.
Are there any hazardous products included in the kit?
The composition of all reagents included in the kit is listed in the package insert. The concentration of reagents is very low; therefore, their use is safe and without any toxicity when handled as described. When requested, DiagnoCure Inc. can provide Material Safety Data Sheets (MSDS) for these particular products along with the kit.
Procedure
Can the laboratory use monolayer liquid-based preparation methods (Thinprep®, Autocyte®, CytoSpin®, etc.)?
No monolayer liquid-based preparation method has been validated by DiagnoCure. It is the responsibility of the laboratory to validate any method other than that which is suggested in ImmunoCyt™/uCyt+™ package insert.
Laboratories may refer to the following publication: “Liquid-based cytology as a tool for performance of uCyt+MC and UroVysion® Multicolour-FISH in the detection of urothelial carcinoma”, C. Mian, M. Lodde, E. Comploj, G. Negri, E. Egarter-Vigl, L. Lusuardi, S. Palermo, M. Marberger, A. Pycha. Cytology 2003, 14: 338-342
Sometimes, the blocking solution does not spread easily over the cell imprint on the slide. What can be done?
The blocking solution (4 drops) should be spread on the slide using a pipette tip by horizontally touching the drops, and avoiding touching the cell surface.
Could the laboratory use product substitutes?
The only recommended product substitutes are:
- Mounting medium: Permafluor™ and Mowiol®
- Harris hematoxylin: Surgipath
- Spray fixative: CytoPrep™
Can we use Gill hematoxylin?
No. It could interfere with the test by overstaining the slides.
How long can ImmunoCyt™ /uCyt+™ slides be kept in the refrigerator?
7 days maximum following the immunoreaction to provide results to the physician. Due to the lack of stability of fluorescence over time, photographs taken with a camera remain the best archival method of ImmunoCyt™/uCyt+™ cases.
Microscopy reading
What are the technical requirements for microscopy reading?
- Fluorescence microscope with mercury lamp (100W)
- 20X and 40X objectives
- Dual filter (Texas Red™ and fluorescein)
Can a microscope with a 60W or 50W mercury lamp be used?
A 100W mercury lamp is recommended for optimal results with ImmunoCyt™/uCyt+™.
Could a rhodamine filter able to read red fluorescence be used on the microscope?
No, a rhodamine filter is not acceptable. The excitation and emission wavelength are slightly different and it could result in lower detection of positive red cells. It is recommended to use specific filters for Texas Red™ and fluorescein.
Could the laboratory use specific filters for Texas Red and fluorescein?
Yes, but the use of a double filter for both markers eases and reduces reading time.
Could regular objectives for standard cytology be used for the ImmunoCyt™/uCyt+™ test?
Yes, but DiagnoCure Inc. recommends the use of specific objectives for fluorescence.
What is the approximate time spent on microscopy reading?
About 20 to 30 minutes per slide at the beginning for a negative slide. Experience brings it to about 10 minutes per slide.
Upon analysis of the positive control, the green and red fluorescence had a low-intensity on the slide. What could be the problem?
First, you should check the microscope adjustment. If the equipment is adequate, you should review the ancillary products used in the technique. Please refer to the Troubleshooting section of the Technical Manual for details or contact DiagnoCure’s Customer Service.
How do we interpret an ImmunoCyt™/uCyt+™ result?
The test is scored:
- POSITIVE: As soon as one confirmed green or red urothelial fluorescent cell is detected. It is recommended that slides with only 1 to 5 positive cells be confirmed by testing a second sample.
- NEGATIVE: If no red or green fluorescent cell is detected on a slide containing more than 500 epithelial cells.
- INADEQUATE: If no red or green fluorescent cell is detected on a slide containing less than 500 epithelial cells
On a negative ImmunoCyt™/uCyt+™ result, how should the 500 required epithelial cells be counted?
An average of one epithelial cell per microscopic field with the 20X objective must be identified. A quick reading of the slide on clear background must be performed to view about 30 microscopic fields on the specimen.
Technical procedure time
How much time is spent on the technical part of the test?
Approximately two hours to prepare 15 slides including 75 minutes for incubation.
In more details:
| Slide identification and preparation of the filter holders | ~10 min |
| Syringe filtration | ~10-15 min |
| Immunoreaction | |
| Pretreatment | ~3 min |
| Blocking solution incubation | ~15 min |
| Antibody solution incubation | ~60 min |
| Rinsing and mounting | ~15 min |
| ~2 hours |