Sample collection for CytoFISH and UroVysion

Over a nine-month period, urine specimens (35-60mL) were freshly collected from a mix of male (ages 36–99) and female (ages 46–91) patients. Cases were not consecutive but based on sufficient sample volume for both UroVysion and CytoFISH testing. The specimens were mixed well with fixative at a ratio of 2:1 (urine:fixative), to prevent bacterial growth and preserve cells (Hologic, Inc., Bedford, MA, USA). Urothelial slides were prepared using the Cytyc ThinPrep® 2000 Processor (Hologic, Inc.) following the manufacturer’s instructions, followed by a fixation process using 4:1 (methanol:acetic acid) Carynoy’s solution for 10 min. Both assays were performed in accordance with the UroVysion procedure, as outlined in the manufacturer’s supplied package insert (Abbott Laboratories, Abbott Park, IL, USA) with the following exception: the probe volume used was 3µLfor UroVysion and 5µL for CytoFISH.

Utilizing Abbott’s VP2000 automated pre-hybridization procedure, the slides were partially digested in pepsin (2500–3000 units/mg; Sigma-Aldrich Co. LLC, St. Louis, MO, USA) at 37°C for 10 min. The cells were fixed for 5 min in 1% formaldehyde solution and rinsed in phosphate-buffered saline (PBS) for 5 min. Slides were dehydrated in 70%, 85%, and 100% alcohol for 1 min each and air dried. Slides were hybridized with 5µL of CytoFISH probe mix (COPY CONTROL 3 Aqua FISH Probe/ COPY CONTROL 7 Orange FISH Probe/ COPY CONTROL 10 Green FISH Probe/ 5p15.2 Red FISH Probe; Biocare Medical, LLC, Concord, CA, USA), and 3µL of Abbott’s UroVysion probe mix. Coverslips were applied and sealed with rubber cement, and placed on the ThermoBrite™ Hybridizer (Abbott Laboratories, Abbott Park, IL, USA); denaturing temperature was set to 74 ± 2°C for 3 min, with hybridization temperature set to 39˚C for 16 h. After hybridization, the slides were removed from the ThermoBrite hybridizer. Rubber cement and coverslips were then removed. To remove unbound or nonspecifically hybridized probes, the slides were washed in 0.4X Saline Sodium Citrate (SSC)/0.3% NP-40 for 2 min at 73° ± 1°C, with gentle agitation every 30 s. This was followed by a second wash in 2X SSC/0.1% NP-40 for 1 min, at room temperature, with gentle agitation every 30 s. The slides were placed in a darkened area on a paper towel and allowed to dry completely. Slides were counterstained with 8µL of 4′,6-diamidino-2-phenylindole dihydrochloride (DAPI), coverslipped and then analyzed with an epifluorescence microscope.

Scoring criteria for CytoFISH Test

Scoring criteria were established following the UroVysion Kit Package Insert (Abbott Laboratories, Abbott Park, IL, USA). The normal cutoff was determined to be three chromosomally abnormal cells maximum and the scoring criteria are:

1.

score at minimum 25 cells with abnormal nuclear morphology;

2.

a positive cell must show multiple chromosomal gains or amplification of 5p15.2 (Fig. 1);

Fig. 1

Examples of (A) normal urothelial cells, (B) urothelial cells with chromosomal gains and 5p15.2 amplifications, and (C) suspicious cases

3.

if ≥ 4 cells show gains for two or more chromosomes in the same cell, the sample is positive;

4.

if < 4 positive cells are found in at least 25 cells, the sample is negative;

5.

if three cells with gains of multiple chromosomes are found, analysis is extended until a fourth positive cell is found, or there are no more cells to score;

6.

if the sample is negative and < 25 cells can be scored, the analysis is invalid.

Scoring criteria for UroVysion Kit

Scoring criteria were established following the UroVysion Kit Package Insert (Abbott Laboratories, Abbott Park, IL, USA). The normal cutoff was determined to be three chromosomally abnormal cells maximum and the scoring criteria are:

1.

score at minimum 25 cells with abnormal nuclear morphology;

2.

a positive cell must show multiple chromosomal gains or loss of both copies of 9p21 locus);

3.

if ≥ 4 cells show gains for two or more chromosomes in the same cell, the sample is positive;

4.

if ≥ 12 cells have zero 9p21 signals, the sample is positive;

5.

if < 4 cells with gains of multiple chromosomes or < 12 cells with homozygous loss of 9p21 are detected in at least 25 cells, the sample is negative;

6.

if three cells with gains of multiple chromosomes or 11 cells with homozygous loss of 9p21 are found, analysis is continued until a fourth multiple chromosome positive cell or 9p21 homozygous loss positive cell is found, or there are no more cells to score;

7.

if the sample is negative and < 25 cells can be scored, the analysis is invalid.

Analytical performance of the CytoFISH probe panel

The analytical performance of the CytoFISH panel was validated based on a broadly applicable preclinical process previously described for hybridizing probes [6]. The CytoFISH panel was validated against normal cells in metaphase from five chromosomally normal individuals to determine analytical specificity and sensitivity. Hybridization was limited to the intended target regions of the 4 probes; no cross-hybridization to other chromosome loci was observed in any of the cells examined.