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Flow cytometric DNA analysis of ulcerative colitis using paraffin-embedded biopsy specimens: comparison with morphology and DNA analysis of fresh samples.

Author(s): Hartmann DP, Montgomery EA, Carr NJ, Gupta PK, Azumi N

Publication: Am J Gastroenterol, 1995, Vol. 90, Page 590-6

PubMed ID: 7717317 PubMed Review Paper? No

Purpose of Paper

Conclusion of Paper

Studies

1. Study Purpose

   This study compared DNA ploidy analysis in matched frozen and formalin-fixed paraffin-embedded (FFPE) specimens of ulcerative colitis and compared the results with morphological analysis. The effects of delayed fixation on DNA ploidy were also investigated. Flow cytometric analysis was conducted on 48 matched FFPE sections and specimens that were stored in 4˚C saline for 30 min or more and then snap-frozen in liquid nitrogen from eight patients with ulcerative colitis, 32 FFPE slides and blocks from 12 patients with ulcerative colitis with dysplasia, and normal colonic mucosa specimens from a single patient fixed in formalin 10 min, 20 min, 30 min, 1 h and 2 h after resection.  Frozen specimens were thawed at room temperature, minced, placed in nuclear staining solution, filtered, and washed multiple times before analysis. Two to three 50µm FFPE sections were deparaffinized in xylene, rehydrated, minced, incubated in acidified saline with pepsin, washed in phosphate buffered saline, filtered, and incubated in nuclear staining solution before analysis. Morphology was determined by histology and classified as negative, indefinite, or positive for dysplasia. 

   Summary of Findings:

   Satisfactory results were obtained from 46 of 48 (96%) frozen specimens and from 40 of 46 (87%) FFPE specimens from the same eight patients. Of these, 33 (72%) of the frozen specimens were aneuploidy with average DNA indices of 1.21 and only three of the FFPE specimens were aneuploid with DNA indices of 1.15, 1.76, and 1.79. Histological analysis of the aneuploid specimens showed active colitis in 20 specimens and inflammation with no evidence of dysplasia. When specimens were analyzed pairwise, 29 pairs were aneuploid in the frozen specimen and diploid in the FFPE specimen, two pairs were diploid in the frozen specimen and aneuploid in the FFPE, one pair was aneuploid in both specimens, and nine pairs were diploid in both specimens.

   Of the 32 specimens from 12 patients with a diagnosis of dysplasia, 16 were histologically negative for dysplasia and were diploid, eight were histologically indefinite for dysplasia and were diploid, six showed low-grade dysplasia histologically and five of these were aneuploid, and the remaining two had high-grade dysplasia and were aneuploid. All specimens from the normal colonic mucosa specimens stored for less than an hour before fixation showed only a diploid peak, but the specimens stored for 1 h or 2 h before fixation also had a small near diploid aneuploid peak (DNA index of 1.11) and this peak was more pronounced in specimens stored for 2 h than 1 h.

   Biospecimens

   • Tissue - Colorectal

   Preservative Types

   • Frozen
   • Formalin

   Diagnoses:

   • Ulcerative Colitis

   Platform:

   Analyte	Technology Platform
   DNA	Light microscopy
   DNA	Flow cytometry

   Pre-analytical Factors:

   Classification	Pre-analytical Factor	Value(s)
   Storage	Time at room temperature	10 min 20 min 30 min 1 h 2 h
   Preaquisition	Diagnosis/ patient condition	Ulcerative colitis with dysplasia Ulcerative colitis without dysplasia
   Biospecimen Preservation	Type of fixation/preservation	Formalin (buffered) Snap frozen

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