Ultrasonic decalcification offers new perspectives for rapid FISH, DNA, and RT-PCR analysis in bone marrow trephines.
The requisite analyses on bone marrow biopsies are increasing: Molecular analyses such as fluorescence in situ hybridization (FISH), polymerase chain reaction (PCR), and reverse transcriptase (RT)-PCR are demanded in addition to morphology and immunohistochemistry to improve diagnostic accuracy. Moreover, analysis of certain molecular prognostic or predictive biomarkers is increasingly mandatory in the assessment of hematologic diseases. In some circumstances, only formalin fixed, bone-containing tissue is available for molecular analysis. Because various fixation and decalcification procedures can impair DNA and RNA quality, there is an urgent need for standardized decalcification protocols which allow FISH and PCR analysis. In this study we developed a routinely applicable decalcification protocol to optimize the molecular analysis method although preserving morphology and immunohistochemical results. Therefore, we compared 2 different approaches including ultrasonic decalcification versus nonultrasonic procedures and ethylenediaminetetraacetate-based reagents versus acid-based ones. In our hands, the combined use of ultrasound and ethylenediaminetetraacetate-based reagents permits successful interphase FISH, PCR, and RT-PCR analysis whereas concomitantly preserving morphology and antigeneicity.
Reineke
Tanja
T
Institute of Surgical Pathology, University Hospital Zurich, Schmelsburgstrasse 12, CH-8091 Zurich, Switzerland.
Jenni
Bettina
B
Abdou
Marie-Therese
MT
Frigerio
Simona
S
Zubler
Petra
P
Moch
Holger
H
Tinguely
Marianne
M
D001706:Biopsy; D001856:Bone Marrow Examination / Q000379:methods; D002118:Calcium / Q000737:chemistry*; D004247:DNA / Q000032:analysis*; D003648:Decalcification Technique; D004492:Edetic Acid / Q000737:chemistry; D005404:Fixatives; D005557:Formaldehyde; D017404:In Situ Hybridization, Fluorescence; D017942:Nitric Acid / Q000737:chemistry; D016612:Paraffin Embedding; D012313:RNA / Q000032:analysis; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods*; D013048:Specimen Handling / Q000379:methods*; D016707:Tissue Fixation; D014465:Ultrasonics
D005404:Fixatives; D005557:Formaldehyde; D017942:Nitric Acid; D012313:RNA; D004247:DNA; D004492:Edetic Acid; D002118:Calcium