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

1. 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

   • Tissue - Bone Marrow

   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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2. 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

   • Tissue - Bone Marrow
   • Tissue - Tonsil
   • Tissue - Lymph Node

   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

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