Optimizing urothelial cell preparation for the human urinary micronucleus assay

Abstract

Biological monitoring of early genotoxic effects in urothelial cells using the urinary micronucleus (MNu) assay is promising for early detection of cancer, such as bladder carcinoma. But many problems are encountered, the major being the poorly differential staining of cells, particularly in women having an important amount of squamous cells. We have optimized the protocol and obtained a differential staining of the cell types present in urine on 10 subjects. Following Carnoy I fixation and Papanicolaou staining, urothelial cells were blue while most squamous cells were pink. This differential staining allowed for optimization of the MNu assay on a single urine void, for both females and males. Even if our MNu means were comparable to the literature, the great variation in reported MNu results could reside in the ability of scorers to distinguish correctly between urothelial and squamous cells. When monitoring exposed populations, this erroneous distinction could largely influence the results, even more in women's urine samples. Given a situation where exposure would not increase micronuclei frequency in vaginal squamous cells, their erroneous analysis in the MNu assay could mask an early genotoxic effect. Therefore, as transitional cell carcinoma of the bladder originates from transformed urothelial cells, restricting micronuclei analysis to urothelial cells could yield a more precise estimate of cancer risk in exposed populations. Moreover, it is hoped that the improvements proposed in this paper will allow for an easier implementation of the MNu assay in various set-ups and enhance its specificity, since MNu are considered a suitable biomarker.

Micronuclei frequency in urothelial cells of bladder cancer patients, as a biomarker of prognosis

“…Regarding the protocol, epithelial cells such as urothelial cells, present cytoplasmic inclusions that can be misclassified when standard staining (i.e., Giemsa) is used. To overcome this problem the use of DNA specific staining has been proposed (Fortin et al, ; Sharma et al, ). We have proved the efficacy of this proposal by using DAPI as the DNA specific staining, which was previously proved for scoring MN in buccal cells of people exposed to arsenic (Martínez et al, ).…”

Associations between Arsenic Species in Exfoliated Urothelial Cells and Prevalence of Diabetes among Residents of Chihuahua, Mexico

“…These differences can probably be explained by different cell counts and cell types present in urine from males and females. The vesical trigone area of an adult female bladder is particularly susceptible to squamous metaplasia ( Fortin et al 2010 ; Tyler 1962 ), a noncancerous change in the epithelial lining that is ultimately responsible for greater numbers of cells in urine from females. Urine from females may also contain vaginal epithelial cells.…”

Estrogen Supports Urothelial Defense Mechanisms

“…Urinary cells were washed and analyzed on a Becton Dickinson FACSCalibur flow cytometer with the CellQuest software (BD Biosciences Immunocytometry Systems). Similarly prepared cell pellets were fixed with Carnoy I fixative and stained by the Papanicolaou technique as described before (56). Most of the cells within the preparations were of epithelial origin.…”