Ultrasonic decalcification offers new perspectives for rapid FISH, DNA, and RT-PCR analysis in bone marrow trephines.
Author(s): Reineke T, Jenni B, Abdou MT, Frigerio S, Zubler P, Moch H, Tinguely M
Publication: Am J Surg Pathol, 2006, Vol. 30, Page 892-6
PubMed ID: 16819333 PubMed Review Paper? No
Purpose of Paper
Conclusion of Paper
Studies
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Study Purpose
The purpose of this study was to determine the effects of decalcification procedures and solutions, as well as fixation duration on the preservation of morphological details and protein in postmortem FFPE bone marrow trephine specimens.
Summary of Findings:
Morphological details were preserved regardless of decalcification solution or procedure. The best morphological preservation was observed in specimens with a fixation time of 24 hours. Ultrasonic decalcification allowed for easier sectioning compared with nonultrasonic decalcification. Immunostaining was better in EDTA-decalcified specimens (USEDECALC or 10% EDTA) than acid-decalcified specimens (USERAPID or 5% nitric acid).
Biospecimens
Preservative Types
- Formalin
Diagnoses:
- Autopsy
Platform:
Analyte Technology Platform Protein Immunohistochemistry Morphology Light microscopy Pre-analytical Factors:
Classification Pre-analytical Factor Value(s) Biospecimen Preservation Time in fixative 12 h
24 h
48 h
1 week
3 weeks
Analyte Extraction and Purification Decalcification solution/ duration USEDECALC
10% EDTA
USERAPID
5% nitric acid
2 h ultrasonic decalcification prior to embedding
3 h ultrasonic decalcification prior to embedding
2 h nonultrasonic decalcification of embedded tissue
Immunohistochemistry Specific Targeted peptide/protein MIB1/Ki-67
CD20
CD3
GlycophorinA
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Study Purpose
The purpose of this study was to determine the effects of decalcification procedures and solutions, as well as fixation duration on the preservation of DNA and RNA in postmortem FFPE bone marrow trephine specimens.
Summary of Findings:
FISH signals in ultrasonic-decalcified bone marrow specimens were comparable to those in noncalcified control lymph node specimens. Acid-decalcification led to higher background and reduced FISH signals. Amplification of longer DNA fragments was inhibited by fixation periods of 1 week, and no PCR amplification was observed after fixation for 3 weeks. Acid-decalcification solutions inhibited amplification of 589 bp DNA fragments, but EDTA-decalcification allowed for amplification of 287 and 589 bp fragments, regardless of whether or not ultrasonic decalcification was used. RT-PCR amplification of a 174 bp Beta-actin fragment was successful, regardless of decalcification solution, however, RT-PCR amplification of a 485 bp fragment was only successful after ultrasonic EDTA-decalcification.
Biospecimens
Preservative Types
- Formalin
Diagnoses:
- Autopsy
- Normal
Platform:
Analyte Technology Platform DNA FISH DNA PCR RNA RT-PCR Pre-analytical Factors:
Classification Pre-analytical Factor Value(s) Biospecimen Preservation Time in fixative 12 h
24 h
48 h
1 week
3 weeks
Analyte Extraction and Purification Decalcification solution/ duration USEDECALC
10% EDTA
USERAPID
5% nitric acid
2 h ultrasonic decalcification prior to embedding
3 h ultrasonic decalcification prior to embedding
2 h nonultrasonic decalcification of embedded tissue
Biospecimen Acquisition Biospecimen location Bone marrow
Tonsil
Lymph node
PCR Specific Targeted nucleic acid Versican
PCR Specific Length of gene fragment 287 bp
589 bp
RT-PCR Specific Targeted nucleic acid Beta-actin
RT-PCR Specific Length of gene fragment 174 bp
485 bp