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Diagnostic Tests

Testing for Ploidy

The technique used to test for ploidy is described in detail in the manuscripts published by Sudbo et al. Figure 1 is a diagram taken from reference 2. It is a flow scheme that represents the technique used in isolating the nuclei and testing for ploidy. This technique cannot be applied to the daily routine of a histotechnologist in a standard histology laboratory. It is a technique that can be learned and reproduced; but it is expensive, time-consuming, and requires special equipment.
Formalin fixed biopsy specimens are processed and embedded in paraffin blocks as described in the Fall 2004 newsletter (under diagnostic tests). For the DNA ploidy studies two 50 m sections are cut and dissociated enzymatically using proteolytic enzymes. This step results in single nucleus isolates which are then spread over a glass slide as a monolayer of single nuclei and stained with Feulgen-Schiff method. The DNA ploidy analysis is performed using the Fairfield DNA Ploidy System. The following criteria were used for the ploidy classification:

  1. The lesion was classified as diploid if only one G0/G1 (2c) peak was present or if the number of nuclei in G2 (4c) peak did not exceed 10% of the total number of epithelial nuclei, or if the number of nuclei with a DNA content >5c did not exceed 1% of the total number of epithelial cells.
  2. A lesion was defined as tetraploid when its G0/G1 (4c) peak was present together with its G2 peak (8c) or when the fraction of nuclei in the tetraploid region exceeded 10% of the total number of nuclei without corresponding S-phase.
  3. A lesion was defined as aneuploid if non-euploid peaks were present or if the number of nuclei with a DNA content >5c or 9c exceeded 1%. The mean coefficient of variation (CV) of the diploid peak for all the 45 cases (diploid, tetraploid and aneupolid) was 5.7%, range 3.3–7.9%. The corresponding numbers for the diploid cases are 4.6%, range 3.6–6.7%. “

Referrences

  1. Sudbø J, Ried T et al. Abnormal DNA content predicts the occurrence of carcinomas in non-dysplastic oral white patches. Oral Oncol. 37 (2001), pp. 558–565. SummaryPlus | Full Text + Links | PDF (384 K)
  2. Sudbø J, Kildal W et al. DNA content as a prognostic marker in patients with oral leukoplakia. N. Engl. J. Med. 344 (2001), pp. 1270–1278. Full Text via CrossRef
  3. Sudbø J, Lippman S et al. The influence of resection and aneuploidy on mortality in oral leukoplakia. N. Engl. J. Med. 350 (2004), pp. 1405–1413. Full Text via CrossRef