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I.

TITLE: BLADDER CANCER FISH ANALYSIS USEING CELLAY PROBES

II. PRINCIPAL: Cellay fluorescence in situ hybridization assay that was developed for the
detection of bladder cancer in urine specimens. It consists of fluorescently labeled DNA
probes to the centromeric regions of chromosomes 3 (gold), 7 (green), and 6 (aqua) and 21
(red). The assay works by detecting urinary cells that have chromosomal abnormalities
consistent with a diagnosis of bladder cancer.

III. SPECIMEN: Slides prepared from fixed cell suspensions and probed.

IV. METERIAL AND EQUIPMENT: cellSens imaging software , Olympus BX61


fluorescent microscope.

V. PROCEDURE: (Before starting scanning of slides, please be properly acquainted with the function of Olympus
BX61 fluorescent microscope)

1. Turn power supply on for mercury light source system. Once the power source has
been turned on, the lamp will need a few minutes to warm up.

2. Turn the microscope power supply on (located under the table).

3. Login to computer and open cellSens imaging software.

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4. To place a slide , open the specimen holder clamping lever and place the specimen
slide on the stage by sliding slide from the front.

5. After placing the slide as far as they will go, gently release the clamping lever

6. Once the slide is properly secured, bring the platform up to operating position using
focus adjustment knob.

7. Turn on the fluorescent light by pressing TSH on the controller

8. Place 20X objective by pressing "20X" button on TSH controller.

9. Change to DAPI filter by pressing buttons FW- to FW+ to cycle through the filter
positions.

10. Using 20X objective locate populated area of cells.


11. Focus cell at 40X magnification.
12. Change to appropriate filters to view color signals.

a. Green: Chromosome 7
b. Red: Chromosome 21
c. Aqua: Chromosome 6
d. Dual Pass (G/R): Chromosome 7/ Chromosome 21.

13. Score signals.

VI. ANALYSIS:

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VII. Quality Control

1. A positive control, i.e., a slide prepared using cells proven by cytogenetic analysis to
contain the abnormality assayed for in the test sample, is processed and analyzed
concurrently with the test sample. In this assay Abbot Molecular control slides are
used. Control slide is prepared with each batch. If control slides fail to meet the slide
acceptance criteria, the assay may not have been performed properly In no case should
test results be reported if assay controls fail.

2. Reasons to Repeat the Assay

The following are situations requiring repeat assays with fresh specimen or existing
slides and the appropriate control slides.

a. If the control slide targets fail to meet the slide acceptance criteria, the specimen slide results are
not reliable.
b. If there are fewer than 100 evaluable nuclei, the test is uninformative and the assay should be
repeated.
c. If, upon assessing the slide quality, any of the technical aspects (signal intensity, background, or
cross-hybridization) are unsatisfactory, the assay must be repeated.

3. Maintenance
Halogen bulb hours are tracked every time the microscope is used.

1. Procedure
a. Flow chart
b. Description of the procedure step by step
c. Data Analysis
d. Data quality assessment
i. Pass criteria
ii. Repeat criteria, technical failure and remedial action
iii. Reporting criteria (positive, negative, no result, QNS)

2. Quality Control
a. Areas
b. Reagents (lot to lot and shipment to shipment). Need to define acceptance and rejection
criteria as well as correction actions for kit and individual reagents
c. Patient results (positive, negative and NT controls). Rotation schedule for positive control
can be provided as an appendix. Failure of control criteria regarding failure of the batch
run

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3. Maintenance (for each equipment: daily/when used, monthly, every 6 month or every year, as
recommended)
4. Assay interference and limitations
5. Troubleshooting
6. References
7. Appendices (with primer sequences, etc…)

FISH Signal Patterns

-Polysomy – Greater than 4 copies on any one channel of probe (green, red, aqua).
Need to have a minimum of four true polysomy cells to call a case positive.

-Tetrasomy – 4 copies on at least two channels, but could be all three.


Need a minimum of ten true tetrasomy cells to call a case positive.

-Trisomy – this happens when you either have 3 copies on all channels or you see the same pattern
repeatedly (single-gain on the same channel for the majority of cells).
Need approximately twenty cells to call a case positive.

-Single-gain – Any cell with additional signals not meeting one of the previous. Mostly these are mixed
pattern cases with an additional copy or two.

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