NIH, National Cancer Institute, Division of Cancer Treatment and Diagnosis (DCTD) NIH - National Institutes of Health National Cancer Institute DCTD - Division of Cancer Treatment and Diagnosis
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Ewing's sarcoma of bone: the detection of specific transcripts in a large, consecutive series of formalin-fixed, decalcified, paraffin-embedded tissue samples using the reverse transcriptase-polymerase chain reaction.

Author(s): Mangham DC, Williams A, McMullan DJ, McClure J, Sumathi VP, Grimer RJ, Davies AM

Publication: Histopathology, 2006, Vol. 48, Page 363-76

PubMed ID: 16487358 PubMed Review Paper? No

Purpose of Paper

The purpose of this paper was to determine if reverse transcription polymerase chain reaction (RT-PCR) analysis for translocation detection of Ewing's sarcoma may successfully be applied in decalcified and non-decalcified formalin-fixed, paraffin-embedded (FFPE) tissue and to determine if differences are present in comparison to fresh or snap frozen specimens.

Conclusion of Paper

The authors report the ability to use RT-PCR to detect translocations in 96% of non-decalcified cases or cases that had been decalcified in formic acid. No differences in results were seen between non-decalcified and formic-acid decalcified biospecimens. Translocations were not amplified in any non-Ewing's sarcoma cases. Decalcification with nitric acid, however, precluded RT-PCR on bone biospecimens. The authors recommend RT-PCR as a diagnostic aid in routine practice.

Studies

  1. Study Purpose

    The purpose of this study was to determine if RT-PCR analysis for translocation detection of Ewing's sarcoma may successfully be applied in decalcified and non-decalcified FFPE tissue and to determine if differences are present in comparison to fresh or snap frozen specimens.

    Summary of Findings:

    RNA quality was adversely affected by formalin fixation and paraffin embedding, as observed by electrophoretic analysis of non-decalcified FFPE specimens in comparison to fresh frozen controls. While RNA isolated from formic acid decalcified FFPE specimens was comparable to RNA isolated from non-decalcified FFPE specimens, nitric acid decalcification resulted in more extensive RNA degradation. RT-PCR analysis of nitric acid decalcified FFPE specimens produced inconsistent results, generating a translocation product in just 16% of Ewing's sarcoma immunohistochemically diagnosed cases, compared to 100% in formic acid decalcified FFPE specimens, 89% in non-decalcified FFPE specimens, and 100% in fresh or fresh frozen specimens. RT-PCR results were confirmed by DNA sequencing of amplicons in a subset of specimens.

    Biospecimens
    Preservative Types
    • Formalin
    • None (Fresh)
    • Frozen
    Diagnoses:
    • Neoplastic - Sarcoma
    Platform:
    AnalyteTechnology Platform
    RNA RT-PCR
    RNA Electrophoresis
    RNA Spectrophotometry
    DNA DNA sequencing
    Pre-analytical Factors:
    ClassificationPre-analytical FactorValue(s)
    Analyte Extraction and Purification Decalcification solution/ duration No decalcification
    5% Formic acid
    5% Nitric acid
    Biospecimen Preservation Type of fixation/preservation Formalin (buffered)
    None (fresh)
    Snap frozen
    View more

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