Measurement of S‐phase fractions in lymphoid tissue comparing fresh versus paraffin‐embedded tissue and 4′,6′ ‐diamidino‐2 phenylindole dihydrochloride versus propidium iodide staining

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Abstract
S‐phase fractions for 62 lymphoid biopsies were calculated, by means of flow cytometry, from both fresh and paraffin‐embedded tissue. The purposes of this study were to determine whether significant differences were seen between S‐phase estimates obtained from fresh and fixed tissue and to compare results obtained with two DNA dyes, namely 4′‐6′‐diamidino‐2 phenylindole dihydrochloride (DAPI) and propidium iodide (PI). The 62 cases consisted of 38 cases of non‐Hodgkin's lymphoma (NHL), 19 reactive samples, and 5 cases of Hodgkin's disease. Fifty‐four of the samples showed DNA diploid profiles. A good agreement between S‐phase results from fresh and fixed tissue was seen, with technical factors accounting for around 20% of the total variance. Using a paired t test, no significant difference was seen between fresh and fixed tissue for diploid cases, but there was a trend for S‐phase estimates from fixed tissue to be higher. If all cases (including the eight DNA aneuploid samples) were included in the analysis this difference just reached statistical significance (p <.05). In a subgroup of 19 of the cases, a comparison was performed on both fresh and fixed tissue of staining with DAPI and PI. A good agreement between results with both DNA stains was found on fresh and fixed tissue, with no significant differences being apparent, and stain‐related factors accounted for only 10% of the total variance.