Summary of Procedure

Key technical considerations

The reagents and instructions supplied in the ImmunoCyt™/uCyt+™ kit have  been designed for optimal performance. By following the correct protocol for ImmunoCyt™/uCyt+™ use, you are able to achieve standardized, reproducible results.

Each kit allows for 50 immunoreactions, including the control slides.

Materials required but not provided

Prior to beginning the ImmunoCyt™/uCyt+™ procedure, ensure that you have all the materials required to run the test.

Sample collection:

• 50% ethanol or isopropyl alcohol to add to an equal volume of urine at the collection site.

Control preparation:

• Micropipette (20µl)
• Tips
• Cytoprep™ fixative (or isopropyl alcohol solution in a spray bottle)

Filtration:

• Syringe and plunger (60ml size)
• Filter holder (for 25mm filter)
• Polycarbonate membrane filters (25mm, 8µm porosity)

Immunocytofluorescence procedure:

• Silanized slides and cover slips
• Staining dishes (9 baths and slide rack)
• 80%, 70% and 50% ethanol
• Deionized water
• Harris hematoxylin without acetic acid
• Glacial acetic acid
• 1X PBS solution with and without polyoxyethylene sorbitan monolaurate (Tween 20™) at 0.05%
• Humid chamber
• Wash bottle
• Permafluor™ mounting medium or Mowiol^®
• Trays for fluorescence slides

Interpretation:

• Well adjusted fluorescence microscope with:
• Mercury lamp (100W)
• 20X and 40X objectives (specific for fluorescence recommended)
• Dual filter (fluorescein & Texas Red™)
• Dark room
• Digital counter
• Table lamp

Miscellaneous:

• Gloves
• Kimwipes^®
• Forceps
• Whatman™ filters grade no.1 and funnel to filter hematoxylin
• Graduated cylinder
• Pipette to measure acetic acid
• Pen with diamond tip
• Parafilm^®

Important note: if you have any difficulties obtaining any of these items, please contact DiagnoCure's Customer Service.

Summary of procedure

	At the collection site Mix an equal volume of 50% ethanol or isopropyl alcohol with the urine sample. At the laboratory Upon reception of the sample at the laboratory, add the ImmunoCyt™ /uCyt+™ fixative solution. Urine samples are kept between 2ºC and 8 ºC for a maximum period of 7 days from the collect date.
	Control slides preparation Apply positive and negative controls cell suspension (included in the kit) on slides and fix with spray.
	Filtration of sample Filter urine using a syringe through a polycarbonate filter mounted on a filter holder. The monolayer liquid-based preparation methods have not been validated by DiagnoCure. It is the responsibility of the laboratory to validate any method other than that which is suggested in the product package insert.
	Cell transfer Remove the filter and place it on a slide. This transfers the cells on the slide; fix with a spray.
	Pretreatment of slides Immerse slides in eight baths (ethanol 80%, 70%, 50%, deionized water and hematoxylin + 4% acetic acid).
	Immunoreaction Incubate slides in humid chamber with: blocking solution for 15 minutes; antibody solution for 60 minutes.
	Mounting Rinse slides in the phosphate buffer solutions. Put one drop of mounting medium on slide and place a cover slip on it. Keep the slides protected from light between 2ºC and 8ºC for maximum period of 7 days until microscope reading.

Interpretation of results / Fluorescence microscopy

Positive control Reference for quality of slides preparation and microscope adjustment (20X)	Negative control Color of background (20X)

Patient slide / Summary

Reading of the slide is done with the 20X objective and confirmation with the 40X objective. Each fluorescent element must be checked on brightfield to confirm the presence of a nucleus (cell).

20X / Reading	40X / Confirmation	Brightfield / Nature of the element

Patient slide / Test results
 

Positive Presence of one confirmed red or green positive cell.  If less than 5 positive cells are counted, it is recommended to evaluate a second sample. Negative Absence of fluorescence on a slide with >500 epithelial cells.  Must obtain an average of one cell per microscopic field with the 20X objective on brightfield Inadequate Absence of fluorescence on a slide with <500 epithelial cells