Preparation of Suspensions of Human Cervical Epithelial-Cells Suitable for Biochemical, Cytochemical and Immunocytochemical Investigations

Slater, T. F.; Beesley, J. S.; Flatman, A; Hammond, R. H.; Jonas, S. K.

doi:10.3892/ijo.2.2.233

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“…In tackling this objective we have used our recently developed method (Slater et al, 1992) G6PD is known to be increased in tumour cells generally (Weber, 1977) and in other dividing cells. Heyden (1974) The data show the number of samples from normal women, and women with CIN 1, 2 or 3, that stained for 6PGD above or below the discriminatory line drawn in Figure 3 (cytochemistry).…”

Section: Discussionmentioning

confidence: 99%

“…The samples were centrifuged and separated on a discontinuous Percoll gradient (Pharmacia) as described in detail previously (Slater et al, 1992). This produced, 1, 2 or 3 bands containing squamous epithelial cells that were collected and washed with PBS.…”

Section: Processingmentioning

confidence: 99%

“…Recently a study on a number of cell lines has supported this further by showing that maximal activities of G6PD and 6PGD in many tumourigenic lines were elevated as compared with normal lines (Board et al, 1990 In the present study interference due to these factors has been avoided by a preparative step that ensures that only cervical epithelial cells are present in the sample material. A suitable method for this purification has been described elsewhere (Slater et al, 1992 …”

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Increased activity of 6-phosphogluconate dehydrogenase and glucose-6-phosphate dehydrogenase in purified cell suspensions and single cells from the uterine cervix in cervical intraepithelial neoplasia

Jonas

Benedetto²,

Flatman³

et al. 1992

Br J Cancer

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The activities of 6-phosphogluconate dehydrogenase and glucose-6-phosphate dehydrogenase have been measured in squamous epithelial cells of the uterine cervix from normal patients and cases of cervical intraepithelial neoplasia (CIN). A biochemical cycling method, which uses only simple equipment and is suited to routine use and to automation, was applied to cells separated by gradient centrifugation. In addition, cells were examined cytochemically, and the intensity of staining in the cytoplasm of single whole cells was measured using computerised microcytospectrophotometry. Twenty per cent of cells in samples from normal patients (n=61) showed staining intensities above an extinction of 0.15 at 540 nm, compared to 71% of cases of CIN 1 (n=14), 91% of cases of CIN 2 (n=11) and 67% of cases of CIN 3 (n=15). The cytochemical data do not allow definitive distinctions to be made between different grades of CIN whereas the biochemical assay applied to cell lysates shows convincing differences between normal samples and cases of CIN. There are no false negatives for CIN 3 (n=14) and CIN 2 (n=10) and 11% false negatives for CIN 1 (n=9) and 14% of false positives for normal cases (n=21). The results of this preliminary study with reference to automation are discussed [corrected]. Images Figure 1

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