Ultrasonic decalcification offers new perspectives for rapid FISH, DNA, and RT-PCR analysis in bone marrow trephines

University of Zurich, Zürich, Zurich, Switzerland
American Journal of Surgical Pathology (Impact Factor: 4.59). 08/2006; 30(7):892-6. DOI: 10.1097/01.pas.0000213282.20166.13
Source: PubMed

ABSTRACT 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.

  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: Decalcification procedures are required in order to prepare histopathological preparations of hard tissues such as bone and teeth. Decalcification is usually performed by immersing the hard tissue in different decalcification fluids with various properties. These decalcification fluids typically include inorganic and organic acids, a neutral fluid containing a chelating agent, or a mixture of solutions. Unfortunately, there is no universal decalcification fluid that satisfies all the requirements of pathologists such as rapid decalcification, easy handling, and minimal tissue damage. Techniques involving use of microwaves (MW) or ultrasonic apparatus (US) have been shown to be useful for shortening the time for decalcification procedures. In the present study, we investigated a unique decalcification procedure that uses a common commercial ultrasonic cleaner and a decalcification fluid (formic acid) containing a free-radical scavenger (D-mannitol). The time required to complete the procedure is approximately half of that required to complete a standard decalcification procedure. In addition, tissue morphology and antigenicity is fairly well preserved after decalcification. The procedure is quick, easy to perform, and achieves decalcification of hard tissue with minimal tissue damage.
    Acta histochemica 06/2014; DOI:10.1016/j.acthis.2014.01.006 · 1.76 Impact Factor
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: Although intraoperative rapid diagnosis is conventionally performed using hematoxylin-eosin (HE)-stained specimens, the use of additional special staining, together with immunostaining techniques, has been examined in recent years to improve diagnostic accuracy. In intraoperative rapid diagnosis, immunostaining should be completed within 7-10 min, because the pathologist is typically presented with an HE-stained specimen within the same time period. We hypothesized that ultrasound may enhance antigen-antibody reactions and reduce the number of immunostaining steps. To clarify the ability of ultrasound to support immunostaining, we first created an ultrasonic generator specifically for immunostaining. Next, we explored the optimal conditions for immunostaining of formalin-fixed specimens to examine the utility of the ultrasonic generator. Finally, we tried immunostaining with the ultrasonic generator using frozen specimens to simulate intraoperative rapid diagnosis. We report herein that ultrasound enables immunostaining of frozen specimens in approximately 10 min.
    Journal of Histochemistry and Cytochemistry 05/2010; 58(5):421-8. DOI:10.1369/jhc.2010.955096 · 2.40 Impact Factor
  • Source


Available from