Disaggregation of normal urothelium and transitional cell carcinoma
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Introduction: Flow cytometry, tumour cell in vivo transplantation, nuclear damage and clonogenic-assay testing rely upon single-cell suspensions derived from solid urothelial specimens, usually derived mechanically or enzymatically. It is essential to adequately disaggregate solid tissue without incurring cell injury. We investigated the optimum method of disaggregating solid specimens of normal urothelium and TCC. Materials and methods: Sixty-nine bladder mucosal and 79 tumour specimens were obtained from 51 patients by 0.5 m L endoscopie biopsies. Eight mechanical and enzymatic disaggregation techniques were evaluated by haemocytometer cell count, a vital dye-exclusion test of cell viability. Glemsa assessment of suspension contamination and a DNA-damage assay to quantify nuclear injury. Results: The mean cell counts were higher for tumour (1.8 × 106. SEM 3.8 × 105) than for normal urothelium (5.4 × 105. SEM 1.8 × 104). Disaggregation with collagenase at a concentration of 200 μg/mL provided the highest (P =0.03) mean cell counts for both normal (5.4 × 105, SEM 2.6 × 104) and tumour (5 105, SEM 3.3 × 104). The mean (SEM) percentage of viable cells in each suspension was 73.7% (3.7%) for normal and 65.6% (7.3%) for tumour. The mean (SEM) viability was highest (P =0.02) using 200 μg/mL collagenase (91%, 3.0). The mean (SEM) suspension contamination was higher for tumour (3.89, 0.46) than for normal (0.42, 0.07). Collagenization provided the lowest sample contamination (P =0.02). The mean (SEM) percentage of cells with detectable DNA damage was 36.7 (10.6) for normal and 47.7% (4.4%) for tumour. The mean DNA injury was lowest (P =0.02) using collagenase (normal 8.9%, SEM 0.56; tumour 10.5%. SEM 0.15). Conclusion: A technique was thus established for disaggregating normal and tumorous solid urothelium using 200 μg/mL collagenase at 37°C for 40 min. This method provides high yields of representative, viable cells with minimally damaged nuclear DNA.
|Number of pages||1|
|Journal||British journal of urology|
|Journal publication date||01 Dec 1997|
|Issue number||SUPPL. 2|
|Publication status||Published - 01 Dec 1997|